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ISSUE 65 MARCH 2022

Atomic Force Microscopy combined with Fluorescence Lifetime Imaging Microscopy (AFM + FLIM) Microscopy approaches in zebrafish RMS Summer Studentship Reports 2021 Microscopy in the Third Age – You are never too old to Learn! At the interdisciplinary heart of the matter: The RMS Engineering and Physical Sciences Committee Plus...News, Calendar, Reviews, Reports 1

ISSUE 65 MARCH 2022

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Atomic Force Microscopy combined with Fluorescence Lifetime Imaging Microscopy (AFM + FLIM)

Sophie Meredith and Peter Adams

18 48 70 88

Microscopy approaches in zebrafish

Edited by Elisabeth Kugler

RMS Summer Studentship Reports 2021

Barbara Altenhuber, Mollie Brown and Elene Lominadze

Microscopy in the Third Age – You are never too old to Learn!

Michael Gibson

At the interdisciplinary heart of the matter: The RMS Engineering and Physical Sciences Committee

Roland Kröger

regulars 14 32 40 64 94 98

Calendar Journal of Microscopy New Member Welcome Office News Company News New Products

reports and other features 36

Darwin’s microscope fetches almost £600,000 at auction

Author Interview: Infectious by Dr. John Tregoning (Niga Nawroly)

Lynne Joyce interview

110 Meet the Staff: Chloe Goode

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infocus is the Magazine of the Royal Microscopical Society (RMS) – the only truly international microscopical society. The RMS is dedicated to advancing science, developing careers and supporting wider understanding of science and microscopy. infocus Magazine 37/38 St Clements Oxford, OX4 1AJ, UK Tel. +44 (0)1865 254760 Email: infocus@rms.org.uk Website: www.infocus.org.uk Scientific Editor Leandro Lemgruber, University of Glasgow, UK Editor Owen Morton Tel + (0)1865 254763, Email: editor@infocus.org.uk Editorial Board Susan Cox, King’s College, London, UK Rebecca Higginson, Loughborough University, UK Ian Titley, Institute of Cancer Research, UK Emily Eden, University College London, UK Laura Fumagalli, University of Manchester, UK Rhiannon Heard, University of Oxford, UK Maadhav Kothari, University of Cranfield and Rolls Royce, UK Advertising Email: advertising@infocus.org.uk ISSN: 1750-4740 © 2021 Royal Microscopical Society infocus is published four times per year by the RMS. Designed and produced by The ImageWorks. Reproduction in whole or in part without permission from the RMS is forbidden. Views expressed in the Magazine are those of the individual contributors and do not necessarily reflect those of the RMS.

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ISSUE 65 MARCH 2022

FROM THE SCIENTIFIC EDITOR Dear Readers, Happy New Year! I hope you all had a relaxing break, enjoying time with family and friends (and pets!). It is with great pleasure that we bring to you our first issue of 2022, with articles covering land and sea. We have an interesting piece from Elisabeth Kugler and colleagues discussing different microscopy techniques and image analysis, and their use in imaging vision development, neuronal circuits and cell fate control in zebrafish – one of the most used model organisms in research. From whole organisms in the sea, we dive into cell membranes. Sophie Meredith and Peter Adams discuss the combined usage of Atomic Force Microscopy and Fluorescence Lifetime Imaging, and how this combination is a great set-up to study the structure, functionality and dynamics of cell membranes. Every year the RMS awards summer studentships for students in their second year (or third year in a four-year degree) of study; giving them the opportunity to carry out projects involving microscopy or image analysis in physical or biological sciences. It is a great way to foster a new generation of microscopists – and luckily for infocus - the awardees write a report at the end of the project. In this issue we publish three of these – Mollie Brown reports the work carried out at University of Strathclyde on creating a pipeline for image analysis of images of pancreas taken using a Mesolens microscope; Elene Lominadze discusses the work at the University of Exeter on image analysis of the movements of larvae of a marine worm; Barbara Altenhuber shows us the work carried out at the University of Leeds, using widefield and confocal microscopy to characterise the distribution of specific proteins in brain tissue. From the young generation to a more experienced one, Michael Gibson gives us striking images and a great account of his involvement with the University of the Third Age. It is wonderful to see how microscopy has helped to bring retired people together - and have fun! And as he says in the title of this article, one is never too old to learn! I do hope this year we can all meet again in person at meetings and congresses. Until then, I take this opportunity to wish you all a great 2022. Slàinte!

Leandro Lemgruber Leandro Lemgruber

COVER IMAGE: Inside a lichen, by Dr Alan Prescott, University of Dundee Mineral crystals from within the heart of a lichen collected from a Birch tree in Dundee.The crystals have been coloured using Photoshop. Scanning Electron Microscope - JEOL JEM 7400F.

Atomic Force Microscopy combined with Fluorescence Lifetime Imaging Microscopy (AFM + FLIM): a powerful

approach to explore the structure and dynamics of biological membranes Authors: Sophie Meredith and Peter Adams Affiliation: School of Physics and Astronomy, University of Leeds, UK Correspondence: p.g.adams@leeds.ac.uk Summary Atomic force microscopy (AFM) and fluorescence lifetime imaging microscopy (FLIM) are two powerful techniques that are often used in the biophysical community as a means of characterising biological membranes. On its own AFM can report on the high-resolution 3-D structure and mechanical properties of biological samples adhered to solid supports. FLIM provides information on the position and local environment of fluorescent pigments within samples. When used in combination, these two methodologies can provide a range of opportunities for investigating biological membranes where the position and local interactions between molecules may result in changes to fluorescence, or, in the example of light-regulated processes, changes to membrane/protein functionality. Here we present an application of combined AFM and FLIM to characterise the structure and photophysics of light-harvesting membranes, as well as using video-speed FLIM measurements to investigate membrane dynamics. This approach may be applied to a wide range of biological samples.

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Introduction to Atomic Force Microscopy and Fluorescence Microscopy Atomic Force Microscopy (AFM) is a powerful technique for recording 3-D maps of surfaces with nanoscale resolution (Santos and Carvalho, 2019). Samples are typically adhered onto a planar support

last decade, with growing recognition that it can be used to observe the structure and mechanics of key biomolecules under relatively natural conditions (Figure 1A). This has included insightful studies of whole cells (Müller and Dufrêne, 2011), lipid membranes (Connell et al., 2013), proteins (Kumar et al., 2017), and polysaccharides (Chen et al., 2016).

surface, such as mica, glass or silicon, and then an

However, AFM has its limitations which include: a

ultra-sharp probe is used to generate a map of

relatively slow image acquisition speed (typically

the differences in height (a topograph). In addition

minutes), a lack of specific chemical recognition

to recording XYZ images, the instrument can be

and a lower resolution than X-ray crystallography

used to measure the mechanical properties of a

or cryo-electron microscopy. Each of these aspects

sample, for example, to see how the elastic modulus

are being addressed in ongoing research around

or deformability of a surface varies in different

the world, e.g., video-speed scanning (Ando, 2019),

locations. The advantages of the AFM technique are:

super-resolution analysis methods (Heath et al.,

a relatively high spatial resolution (~1 nm laterally

2021), and the use of functionalised AFM probes for

and ~0.1 nm in the Z-plane), the excellent signal-to-

chemical recognition (Vasilev et al., 2019) but these

noise (so averaging is not required) and the ability

approaches are not straightforward. Fluorescence

to assess samples in a hydrated state and in real

microscopy can be a complementary tool that

time (so sample fixation or freezing is not required).

offsets some of these limitations (Figure 1B).

These features may explain why AFM has seen

Standard fluorescence microscopes can record

increasing use in the biological sciences over the

images very quickly (typically seconds) and allow

Figure 1. Comparison of AFM and FLIM methodologies. (A) Schematic of part of the Atomic Force Microscopy instrument. A sharp probe at the end of a flexible cantilever is scanned across the surface of a sample.Topographs (height maps) can be generated with nanometre-resolution. (B) Schematic showing laser excitation of a sample (a tightly-focussed laser spot might be 500 nm in diameter). If the sample contains pigments which can accept the energy then this causes fluorescence (emission of photons) which can be detected by a fluorescence microscope. (C) A cartoon of the principle behind Förster Resonance Energy Transfer (FRET). (D) Schematic of combined AFM and FLIM. (E) An example AFM topograph. (F) An example FLIM image, where each pixel has both a saturation (dark-to-bright) and a false colour (blue-to-red) scale representing the fluorescence intensity and lifetime, respectively.

us to observe dynamic biological processes, such

burst then we observe a quantifiable fluorescence

as molecular diffusion or assembly processes. They

increase as the carboxyfluorescein is released

can also verify the position of specific molecules

and becomes de-quenched. Thus fluorescence

because only the fluorescently-tagged (or naturally

quenching (via FRET or concentration-quenching)

fluorescent) molecules will provide the fluorescence

can be a useful tool for various experiments related

signal. Therefore, it can be a good strategy to use

to molecular interactions.

AFM and fluorescence microscopy in parallel as their strengths offset their weaknesses.

Fluorescence analysis using FRET and FLIM (Förster Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy) The fluorescence properties of pigments are sensitive to their local environment (e.g. pH, temperature, or the proximity of other molecules), and so fluorescence methods can also be exploited to reveal addition information beyond the structure of samples. A phenomenon known as Förster Resonance Energy Transfer (FRET), in which a donor pigment and an acceptor pigment may interact if they are spectrally (i.e., energetically) similar, is commonly used to assess protein-protein interactions or molecular distances. Energy transfer will occur when the donor and acceptors are in close proximity (Figure 1C) and causes the donor fluorescence intensity to reduce, termed donor “quenching”, and the acceptor fluorescence intensity to increase due to receiving additional energy. The average distance between donor and acceptor molecules can be calculated very accurately based on Förster theory if these distances range from 1-10 nanometres, leading some researchers to refer to FRET as a “molecular ruler” (Roy et al., 2008). Alternatively, certain pigments are known to “selfquench” each other in a concentration-dependent manner and we can exploit this in experiments

Quenching can be quantified via changes to the fluorescence intensity but the disadvantage here is that it can be challenging to distinguish between a reduced fluorescence caused by FRET or simply a decrease in the concentration of the fluorophore. Alternatively, FRET and other types of quenching are also manifested as an increased rate of decay by fluorescence because the excited electronic states of donor molecules now have additional pathways for decay (e.g., both transfer and fluorescence). This can be measured with time-resolved fluorescence (TRF) spectroscopy, which employs specialised detectors and timing electronics that act as a picosecond-accuracy stopwatch to measure the length of time between photon absorption and re-emission by fluorescence. Measuring millions of single-photon events in this manner allows us to calculate the “fluorescence lifetime” of a pigment which, in turn, can be used to quantify the quenching. Thus, a decreased fluorescence lifetime will reveal FRET independently of the pigment concentration and often more accurately than the fluorescence intensity change. Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique which acquires fluorescence images where every pixel contains both the intensity and lifetime information (Figure 1F) (Trautmann et al., 2013). FLIM has been used to determine how molecular interactions vary across an image, for example, proteins interacting in different parts of a cancer cell (Provenzano et al., 2009).

where a change in molecular concentration tells us

This article looks at the opportunities offered by

something about the system of interest. For example,

using AFM and FLIM in correlation. This includes

we can assess the stability of polymeric membranes

taking images on separate samples and correlating

that are designed to encapsulate drugs by including

two different datasets and the more challenging task

self-quenching carboxyfluorescein dye molecules

of spatially-correlated AFM+FLIM (Figure 1D-F).

(Seneviratne et al., 2020). If the polymer membranes

The complementarity of the nanoscale resolution

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of AFM and the ability to quantify changes in

membranes are packed full of “light-harvesting” (LH)

photophysical properties by FLIM is an enticing

and “photosystem” (PS) membrane proteins which

combination.

contain chlorophyll and other pigments for energy

Case study: understanding photosynthesis using AFM and FLIM

absorption and transfer (Johnson et al., 2014). However, there are still gaps in our knowledge of this important process. Specifically, we do not know the precise photophysical (energy transfer)

Our research group is interested in understanding

pathways and their timescales of occurrence within

the molecular mechanisms of photosynthesis in

individual LH proteins. There are also questions

plants and other organisms. In plants, the primary

about how the overall membrane system adapts to

reactions of photosynthesis take place within

changes in light intensity. Recent research suggests

organelles called chloroplasts. Contained within the

that exposure to elevated light intensities can

chloroplast is a complex system of bio-membranes

trigger dynamic rearrangements of LH proteins

called “thylakoids” (Figure 2A), which are the

and can trigger changes in the energetic balance of

location of solar energy absorption, energy transfer

the system (Johnson and Wientjes, 2020). One way

and the initial conversion of energy to a chemical

to understand more about the structure of these

state. Decades of research have revealed the overall

systems is using AFM and FLIM.

architecture of the thylakoid membranes, atomic

In recent research, we correlated the optical and

resolution structures of the proteins embedded

structural properties of thylakoids extracted

within these membranes, and information about

from plants by a combination of FLIM and AFM

the bioenergetic pathways (Blankenship, 2021). We

measurements (Meredith et al., 2021). We obtained

know that thylakoids are made up of interconnected

FLIM images of extracted thylakoids adhered to

membranes

disc-like

glass coverslips, where the fluorescence signal is

arrangements, called grana, and single layered

from the natural fluorescence of chlorophylls that

regions, called stromal lamellae. The thylakoid

are within LH proteins. These images show that

which

form

stacked

Figure 2. Investigation of the thylakoid membranes extracted from chloroplasts. (A) Cartoon of a chloroplast. (B) FLIM image of extracted thylakoid membranes adhered onto a hydrophilic glass surface. (C) Normalised fluorescence decay curves showing data from thylakoids (blue) versus isolated LH proteins (red). (D) AFM image of a similar sample as in (B).The topograph shows small, adhered membrane patches (ringed green) and also larger multilayered structures (ringed blue). (E) A zoomed-in topograph of a multilayered thylakoid extract. (F) A height profile drawn along the green line in panel (E), showing the multilayer steps of the membrane. Panel (A) image credit:Wikimedia Commons (public domain). Panels (B)-(F) are adapted from Meredith et al. 2021, and is used and licensed under CC BY 4.0.

thylakoids appear as distinct objects, small spots of

magnification AFM images (Figure 2E) show

fluorescence often below 1 micron in size (Figure

multilayered structures and we can draw profiles

2B). These fluorescent spots all appear to have

(Figure 2F) to assess the increase in height over

similar, quite short fluorescence lifetimes of ≈0.5 ns

consecutive multilayers, finding stacks of ≈300 nm in

(blue). Note, all FLIM images have a colour scale with

height. LH and PS proteins are known to be 5-10 nm

fluorescence lifetime represented from blue (short

in height, so it is likely that even the smallest objects

lifetime) to red (long lifetime) and an intensity scale

observed via AFM must consist of a few stacked

representing the total counts in each pixel. We can

protein-rich membranes, increasing up to tens of

quantify the fluorescence quenching by extracting

stacked membranes for the largest objects. The

the TRF data from the bright pixels within this FLIM

structures observed by AFM are consistent with the

image. The graph generated (Figure 2C) shows

tightly stacked thylakoid membranes observed in vivo

the exponential decay of the fluorescence signal

(e.g., in intact membranes by electron microscopy).

that allows us to quantify a fluorescence lifetime

The short fluorescence lifetime suggests that the

of 0.4 ns. AFM images reveal that these objects

LH proteins are in a “self-quenched” state. Together,

have a heterogeneous size distribution. In a low-

this is a correlation of the nanoscale structure and

magnification field of view (Figure 2D), a variety

fluorescence of these important biomembranes.

of structures are observed from relatively compact

The fact that such a heterogeneous and disordered

assemblies (100–200 nm laterally and 10–100 nm in

distribution of randomly adhered membranes is

height, ringed green) to large microscale structures

found highlights the need for a method to promote

which contain distinct multilayers (3–4 μm laterally

the formation of large, homogeneous membrane

and up to 750 nm in height, ringed blue). Higher-

structures that are suitable for quantitative studies.

Figure 3. Microscale template patterns on glass coverslips for microscopy. (A) A schematic of the process of UV photolithography. By use of a photomask we induce crosslinking of special photo-active lipids in micro-patterns, in order to generate a grid-like pattern of the polmerized membranes.This acts as a template into which biological membranes will readily fuse. (B) The special lipids (DiynePC) used for photo-crosslinking into a stable form. (C) An example FLIM image of an empty polymerised template.This image represents an 80 × 80 µm region.This figure is adapted from Meredith et al. 2021, and is used and licensed under CC BY 4.0.

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Figure 4. Analysis of “hybrid membranes” by correlated FLIM and AFM measurements. For (A) and (B): the left panel is an AFM topograph, the centre panel is the “Template FLIM channel” and the right panel is the “chlorophyll FLIM channel”. (A) Correlated FLIM+AFM data showing a single square of the polymerised lipid “empty” template. (B) Correlated FLIM+AFM data showing a similar region as in (A), but after the corrals were “backfilled” with the extracted thylakoids and synthetic lipid vesicles to form the hybrid membrane. (C),(D) Profiles drawn across the region in (A) or (B), respectively, showing the AFM height (red), FLIM intensity from the template (green), and FLIM intensity from chlorophyll (blue).This figure is adapted from Meredith et al. 2021, and is used and licensed under CC BY 4.0.

A new micro-patterned template for assessing biomembranes Next, we collaborated with a research team in Japan who produce microscale templates on glass surfaces that are suitable for stabilising biomembranes for more detailed FLIM/AFM studies (Yoneda et al., 2020). These templates take the form of a 2-D array pattern, comprised of empty boxes of 20 × 20 µm in size, for the membranes to adhere (Figure 3). Now, we can take our complex biological membrane of choice and deposit it into the template pattern. Natural membranes will fuse with the exposed edges of the template, so long as they are mixed with synthetic lipid membranes. It appears that the synthetic lipids support fusion and rearrangement of the membranes into a single layer that is confined to the corral (box) region. There are multiple benefits of generating a micro-array pattern of biomembranes, particularly, that they provide an obvious target in the coral regions and a stable, planar form of membrane that is amenable to microscopy.

2021). Extracted thylakoid membranes and synthetic lipid vesicles were incubated with a template pattern and after 20 minutes a stable “hybrid membrane” was generated. To determine the hybrid membrane structure and correlate this to the fluorescence properties, an instrument combining AFM with FLIM was used to record nanoscale topography maps spatially correlated to multi-channel fluorescence data. The AFM image and height profiles (Figure 4A and 4C red line) revealed that the “empty” template pattern of polymerised lipids was the expected ~4.8 nm in height and this aligned with the fluorescence intensity of the template from the partner FLIM image (Figure 4C green line). After the formation of the hybrid membranes, FLIM images show that there was largely homogeneous chlorophyll fluorescence within the square corral regions with no resolvable defects at this scale (Figure 4B). The increase in the chlorophyll fluorescence intensity (Figure 4D blue line) corresponded with a small change in the AFM height of just 0.2 nm (Figure 4D red line). In summary, the FLIM images revealed clear array patterns where the vast majority of chlorophyll fluorescence is localised

To validate this approach and learn more about

within the square corral regions defined by the

photosynthesis, we utilised the same thylakoid

template. These patterned hybrid membranes were

membranes described in the last section to produce

highly reproducible, with similar dimensions and

micro-array patterned membranes (Meredith et al.,

fluorescence intensity across multiple preparations.

Figure 5. Analysis of the dynamic assembly processes occurring during hybrid membrane formation. (A) Time-lapse series of FLIM images during the formation of hybrid membranes. Each panel shows a 20 s acquisition during the real-time membrane assembly. (B) Analysis of the kinetics of hybrid membrane assembly as tracked by the fluorescence signal from lipids (red), photosynthetic proteins accumulating into hybrid membranes (green), and extracted thylakoids which adhere non-specifically (blue). (C) Cartoon of the self-assembly of hybrid membranes.This figure is adapted from Meredith et al. 2021, and is used and licensed under CC BY 4.0.

AFM topographs reveal that the membrane

proteins in a diluted form compared to natural

structure is mostly flat and homogeneous across

membranes but, yet, maintains their activity for

widespread areas. Thus, the average measured

further analysis.

thickness of the hybrid membrane was inferred to

Observing dynamic changes with FLIM: watching membrane assembly

be 4.6 nm. The precise spatial correlation between chlorophyll fluorescence and the topography of the deposited membrane demonstrates that the LH and PS proteins are present specifically within the corral regions.

possible to acquire images in rapid succession and to acquire “video-speed” images to investigate changes

But what do we learn about the biological system?

to samples in real time. For our photosynthetic

Again, the FLIM data reveals important information

model, we wanted to watch hybrid membranes

about the photophysical behaviour, which informs

assembling in order to increase our understanding

us about the energy balance of the LH proteins. In

of the changes in photophysics of the incorporated

these hybrid membranes, we find that the average

proteins and the occurrence of micro/nanoscale

fluorescence lifetime of the chlorophyll signal is

topographical features. In a biophysical context,

approx. 4.0 ns, which is similar to the value known

one may also wish to understand more about the

to represent isolated LH proteins in an entirely

inherent diffusivity of these proteins. Therefore,

“non-quenched” state. This long lifetime is in stark

the kinetics of protein insertion into the hybrid

contrast to the short lifetime found for thylakoids

membranes were studied in real time by acquiring

(0.5 ns). The long average lifetime suggests that

a time-lapse series of FLIM images (Figure 5A)

the proteins became spaced-out within hybrid

(Meredith et al., 2021).

membranes so that the protein-protein interactions found in the natural thylakoids are relatively rare. It is interesting that this nanotechnological platform appears to present the natural photosynthetic

FLIM measurements have the advantage that it is

ISSUE 65 MARCH 2022

Inside corral regions, there was a gradual accumulation

chlorophyll

fluorescence

characterised by a predominantly long fluorescence

lifetime (Figure 5A, red colour). Whereas, across

system and, in future, may allow us to perform

the image various globular particles with short

measurements of low-concentration LH proteins

fluorescence lifetimes became more numerous

diffusing within these corrals. In the process of

over time (Figure 5A, large blue/green spots),

this research, we are improving our understanding

presumably representing extracted thylakoids that

of the molecular basis of photosynthesis and the

had not merged with the synthetic lipid bilayers.

potential for nanotechnology that exploits light

We found that lipids assemble most quickly, with

harvesting components.

a more gradual accumulation of LH proteins, and that both eventually saturate as the corral area was filled (red vs green curve, Figure 5B). Whereas, non-specifically adhered thylakoid membranes continued to slowly adhere over time (blue line, Figure 5B). Considering the kinetic and visual data on the assembly process overall, we hypothesised that extracted thylakoids adhere on top of the nascent lipid bilayer and act as reservoirs from which photosynthetic proteins undergo diffusion down a concentration gradient into the spreading hybrid membrane, as proposed in the cartoon in Figure 5C. Random Brownian motion in 2-D is expected to lead to an overall migration of membrane proteins from a high concentration in the thylakoids to a lower concentration in the hybrid membranes, tending toward a lower energy state of dynamic equilibrium. Watching this dynamic assembly process is interesting from a photosynthesis viewpoint as it bears similarities to the dynamic protein rearrangements which may occur in natural thylakoids (Johnson and Wientjes, 2020). We imagine that other researchers could use time-lapse FLIM measurements to obtain

For other biological systems, we anticipate that the combination of FLIM and AFM could be used correlate changes in the conformation of membrane proteins to their arrangements. FLIM could be useful for researchers who already perform FRET measurements on their own biomolecules-ofinterest (i.e., a protein that is tagged with FRET donor and acceptor pigments). One could imagine various different experimental configurations, for example, FLIM-FRET assessment of (i) single proteins tethered onto glass coverslips, (ii) protein-protein or protein-lipid interactions for transmembrane proteins within surface-supported lipid bilayers, or (iii) various tagged biomolecules within whole cells. AFM can then provide complementary structural information, for example, of (i) single-protein positions, (iii) nanoscale protein arrangements within a membrane, or (iii) cellular mechanics. Micro-array patterns have their own applications, for example, in the development of biosensors (Bally et al., 2010) where pattern-recognition technology may allow more streamlined and automated computer-based analysis.

interesting information about the assembly of

Acknowledgements

alternative biomembranes and the migration of

The collaboration between Leeds and Kobe

other fluorescent biomolecules.

was supported by an International Exchanges

Concluding remarks

Cost Share award from The Royal Society UK

Overall, the use of FLIM allowed us to quantify the chlorophyll fluorescence decay rates towards understanding more about the energy transfer and energy dissipative properties of LH proteins, whilst AFM provided nanoscale spatial information about the membrane structures. These micro-array patterns allowed us to assess a natural mixture of LH and PS proteins the within a model membrane

(IEC/R3/183029). Meredith was supported by a Biotechnology and Biological Sciences Research Council (BBSRC, UK) studentship (BB/M011151/1). Adams was supported by Engineering and Physical Sciences Research Council (EPSRC, UK) grant (EP/ T013958/1). The PicoQuant FLIM+AFM instrument at Leeds was acquired with funding from the BBSRC (BB/R000174/1).

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Calendar Due to the ongoing impact of Covid-19, it is anticipated that forthcoming RMS events may continue to be affected. We are hoping to make a long-desired return to in-person events this year, although we will continue to organise a number of virtual events. The following information was correct at the time infocus went to print but could potentially be subject to change in the coming weeks. Please visit our event calendar at www.rms.org.uk for the latest updates. Our online calendar includes all the details about forthcoming talks in the International Microscopy Lecture Series – a joint, online initiative established between the RMS, and a number of international societies. You can also sign up for the popular Imaging ONEWORLD talks covering all aspects of microscopy and imaging. These take place on Mondays at 1pm (GMT). If you have any questions about a booking you have already made for an event, or need any help or advice, please contact us at info@rms.org.uk

2022 March 7 – 10 Virtual Flow Cytometry Data Analysis Course Spring 2022

April

July 5–6

Frontiers in BioImaging 2022 – Birmingham, UK

20 – 22 FlowcytometryUK 2022 – Birmingham, UK

September

12 – 13 Virtual EBSD 2022

11 – 15 Abercrombie Meeting 2022 – Oxford, UK 12 – 16 Flow Cytometry Course 2022 – York, UK

June

6 – 10 7 – 10

Cryo-Electron Microscopy Course 2022 – Harpenden, UK elmi2022 - Logomo, Turku, Finland (Non-RMS Event)

Microscopy: Advances, Innovation, Impact 2022 – incorporating RMS AGM & Section AGMs – London UK

For further information on all these events, please visit our Event Calendar at www.rms.org.uk

ISSUE 65 MARCH 2022

Featured RMS events Virtual EBSD 2022 12 – 13 April (Online) Scientific Organisers: Dr Ben Britton (University of British Columbia / Imperial College London); Professor João Quinta da Fonseca (University of Manchester); Dr Katharina Marquardt (Imperial College London) The EBSD 2022 meeting will be held in a virtual format. The meeting and workshops will be live, with the main meeting on Tuesday 12 and Wednesday 13 April 2022 at 13:00 BST/08:00 EDT/14:00 CEST/05:00 PDT. We have selected this format to encourage participation from the global electron backscatter diffraction (EBSD) community and to support engagement from a wide range of participants, as well as reflecting on the on-going challenges related to the global COVID-19 pandemic. Our two “half-day” meeting will encourage sharing the latest developments and applications of EBSD-related microscopy methods. In this virtual format, we will also encourage clusters of researchers to independently register for the meeting, but where reasonable to host

cluster-viewing at their host institution (more details about this format will be provided during registration). The Annual UK-based EBSD meeting is an opportunity for the EBSD community to meet and share new developments and applications of EBSD, as well as related techniques that are commonly used to explore samples and materials within the geoscience, materials science & engineering, physics, and emerging applications from the biological communities. Talks will likely include state-of-the-art developments in instrumentation, new software developments, new techniques, as well as applications and use of EBSD, transmission Kikuchi diffraction (TKD), electron channelling contrast imaging (ECCI), and related microscopy modalities. As part of this series, we continue to be excited to hear from those who use these techniques to further the understanding of applied science and engineering challenges, as well as industrial challenges (including the use of EBSD data in Industry 4.0). www.rms.org.uk

Cryo-Electron Microscopy Course 2022 6 – 10 June, Harpenden, UK Scientific Organisers: Dr Eudri Venter (Rothamsted Research) The popular Cryo Electron Microscopy course will return for 2022, with additional new content not included previously. Covering both theoretical and practical aspects of sample preparation and cryo EM techniques, this residential course is ideal for anyone new to cryo EM, but can also be attended by those wanting to refresh their existing skills. This intensive five day course sees numerous topic experts covering a variety of sample preparation techniques (including high pressure freezing, rapid cooling and cryo sectioning), as well as cryo-microscopy operation for both scanning and transmission EMs (including specialist Frontiers in BioImaging 2022 5 – 6 July, Birmingham, UK Scientific Organisers: Dr Joelle Goulding (University of Nottingham); Dr Martin Jones (Francis Crick Institute); Dr Deirdre Kavanagh (University of Oxford); Dr Leandro Lemgruber (University of Glasgow); Dr Ferran Valderrama (St Geroge’s University) Frontiers in Bioimaging 2022 will focus on the latest developments in optical and electron microscopy as well as image analysis. Sessions will cover novel technical developments and applications of these microscopy-based approaches to key cell and molecular biology FlowcytometryUK 2022 20-22 July, Birmingham, UK Scientific Organisers: Derek Davies (Francis Crick Institute); Andy Filby (University of Newcastle); Rachael Walker (Babraham Institute) This meeting will consist of themed plenary sessions with talks from invited speakers. There will also be parallel scientific workshops organised by members of the cytometry community and parallel commercial workshops. There will be a large exhibition and the opportunity to network with flow and image cytometrists from all over Europe and beyond. The meeting will highlight advances in flow and image instrumentation, high content screening, cancer and stem cell biology, applications of clinical cytometry and the development of novel probes and approaches in many areas of biomedical research. www.rms.org.uk

ISSUE 65 MARCH 2022

techniques such as freeze fracturing and cryoSTEM tomography). Each of the topics will include tutorials and demonstrations, although most the time will go into hands-on practical sessions. The course is aimed at life sciences, however the techniques can be applied to other fields, and we therefore invite participants from any area of academia or industry to attend. We also recommend that participants bring their own samples along, as a large portion of the course is dedicated to giving attendees the opportunity to apply the taught techniques to own research. Attendees will be required to wear appropriate PPE and provide evidence of negative Covid status before coming on to the Rothamsted Research site (international travellers will be required to adhere to the most recent uk.gov guidelines at the time of travelling). www.rms.org.uk questions with an overarching aim to bring insights on how they participate in our understanding of human health and disease. We aim to provide an environment where early-careers and established researchers can meet and engage with a broad range of imaging approaches and to make valuable contacts with leading groups in the field. We will be accepting abstracts for both oral and poster contributions. This event will also have opportunities for companies to exhibit and sponsor. www.rms.org.uk

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ISSUE 65 MARCH 2022

Microscopy approaches in zebrafish

By Nathalie Jurisch-Yaksi, Agnese Kocere, Elisabeth Kugler, Ryan B. MacDonald, Christian Mosimann, and Emre Yaksi * Authorship in alphabetical order by surname Edited by Elisabeth Kugler (University College London) Correspondence: Elisabeth Kugler kugler.elisabeth@gmail.com In many scientific fields, research is reliant on microscopy to acquire, describe, and model fundamental processes. With this need for microscopy and multitudes of microscopy techniques existing, it can be challenging to identify the most suitable technique for a specific purpose. We here examine microscopy techniques to study cell fate control, vision development, and neuronal circuits covering various temporal, spatial, and computational scales in zebrafish.

Zebrafish has become a pivotal model organism

enabled three-dimensional image acquisition of

across fields, providing new insights into our

complex samples over time, making 4D-timelapse

understanding from the cytoskeleton, over cells,

in multiple colours to capture developmental

to tissues, and organs. Characteristics such as high

processes accessible [2]–[6].

genomic similarity to humans and experimental accessibility to genetic manipulation allow indepth studies and novel insights. However, the true strengths of zebrafish as model organism

individual planes of a translucent sample, generating a sequence of optical sections that assemble to a 3D representation [3]. Illumination and acquisition

are that they develop ex utero, allowing them to

are uncoupled and parallelised, enabling multi-lens

be studied from the one-cell stage onwards, and

setups to capture a sample suspended between

that they are transparent, enabling non-invasive

lenses that can be rotated for 360-degree

imaging from heartbeat to brain development [1].

acquisition. In addition to capturing sample volume,

Combining these features with a range of transgenic

the rapid scanning and distributed laser power result

reporter lines has opened previously inconceivable

in significantly lower phototoxicity than confocal

opportunities to answer fundamental biological

scanning microscopy, rendering LSFM well-suited

questions and push knowledge boundaries.

to live-image developing zebrafish embryos with

Due to these traits, the main type of microscopy

fluorescent reporters in toto. Consequently, LSFM

used in zebrafish studies is light microscopy, mostly fluorescence-based and in more than two dimensions (2D). The produced microscopy data are highly complex with up to 6D, including space

of zebrafish embryos has revealed unprecedented details of vertebrate development, including details of endoderm migration, neuronal precursor paths, and heart tube formation [5]–[9].

(x,y,z), wavelengths, time-points, and multi-points.

Through an active, open-source-embracing LSFM

Hence, a critical consideration is data handling and

community, the steps and components to build a

computational analysis to complement studies and

growing variety of LSFMs have come into reach of

reinform image acquisition, allowing truly data-

interested labs worldwide. Initiatives like OpenSPIM

driven microscopy.

(openspim.org) provide comprehensive parts lists,

We here examine the role of microscopy to study cell fate control, vision development, and neuronal circuits in zebrafish covering various temporal, spatial, and computational scales.

Light-sheet fluorescence microscopy to study early development by Christiann Mosimann and Agnese Kocere (CU Anschutz, USA)

LSFM applies a sheet of laser light that scans

assembly instructions, and a growing user base to seek support [10], [11], [12]. Yet, pragmatically speaking, the complex optical assembly and maintenance of a custom LSFM setup are out of reach for most biology-focused researchers who wish to utilise rather than develop LSFM [10]: acquisition of the required parts is not trivial, several components require a precision workshop, and the entire build can take months from beginning to completion. Nonetheless, several commercial plugand-play setups are available, with variable degrees

Even though zebrafish are a prime subject to

of specialisations for select applications of imaging

study early development, capturing the complex

fixed or live cells, tissues, organoids, or embryos.

coordination that forms the entire embryo in

To bring LSFM to the masses, the Flamingo LSFM

development has remained challenging due to the

(www.involv3d.org) can be shipped and loaned to

embryo’s spherical geometry. The introduction of

interested labs for individual projects (currently

light-sheet fluorescence microscopy (LSFM) has

USA only), reducing the limited access to LSFM

ISSUE 65 MARCH 2022

Figure 1.1.Through the looking glass: four-dimensional imaging of transgenic zebrafish embryos using the Zeiss Z.1 light sheet setup (left, with illuminated sample chamber). Engineered transgenic zebrafish expressing fluorescent reporters marking the notochord (middle panel, magenta) and hindbrain regions (magenta and green) at 24 hours of development, and the lateral plate mesoderm (right panel, magenta) and the progenitor cells forming the cardiovascular lineages (green) at 12 hours of development.

hardware to shipping costs and desk space.

and subsequent projections of spherical embryo

Our lab focuses on decoding the mechanisms of cardiovascular cell fate emergence from uncommitted

lateral

plate

mesoderm

data [5] also allow us to chart the emerging lateral plate mesoderm before it partitions into the heart, blood vessels, blood, and additional lineages [14].

development and congenital disease [13]. We

Even though a significant tool in the arsenal of

generate and apply novel transgenic zebrafish

developmental biologists and zebrafish researchers,

expressing fluorescent proteins under the control of

LSFM remains a complex application. Beyond

gene-regulatory elements active in early progenitor

challenging sample mounting and accessibility to

cells. Once established through transgenesis,

fluorescent reporters that highlight the desired

fluorescent reporters let us observe previously

biological process, LSFM routinely generates

inaccessible developmental processes and cell types

exceedingly large datasets: multi-colour 4D imaging

live in blue, green, or red fluorescence.

of one embryo (full capture for each channel at

Mere LSFM users, we have been an early adopter of the commercial Zeiss Lightsheet Z.1 microscope that combines three lenses (two for illumination, one detection) with a climate-controlled sample

several angles every few minutes over hours) results in high triple-digit Gigabytes of imaging data – an ordinary desktop PC cannot handle the subsequent multiview alignment and post-processing [10]. Consequently, routine LSFM data assembly and

chamber in a user-friendly box.With interchangeable

output demand a significant investment into

detection lenses ranging from 10x to 63x, the Z.1

computational processing power, including a suite of

offers a versatile imaging platform for a variety of

software solutions to tackle individual steps ranging

samples, but especially for developing zebrafish:

from open-source, such as ImageJ/Fiji-based plugins

suspended in agarose or in optically clear Fluorinated

[15]–[17] or dedicated multiview packages to

ethylene-propylene (FEP) tubing, we routinely image

commercial programs such as Zeiss’ ZEN, or Imaris.

gastrulation-stage embryos (5.3 hours) as well as

Training of new lab members in LSFM operation and

one-to-two day old zebrafish in toto, still or as time-

analysis is a considerable endeavour that, due to its

lapse. Imaging transgenic reporter embryos, we

complexity, often takes longer than introductions

captured the continuous formation of the zebrafish

to a conventional confocal microscope. While we

heart’s ventricle, adding to previous observations

all dream of big datasets, pragmatically speaking,

based on imaging of fixed embryos [7]. 4D imaging

individual lab members usually work with one-to-

Figure 2.1.Visualisation of small-scale protrusions in MG in a 5-day old fish (scale bar 10µm).

three 4D datasets at a time and in series. While

the expansion of established fluorescent reporter

seemingly few, the processing time for each dataset,

transgenics, new staining methods, and increasingly

even without downstream analyses such as reporter

accessible LSFM setups and expertise, zebrafish

co-expression, cell tracking, or morphometric

researchers have more opportunities than ever to

analysis, occupies an imaging workstation for the

illuminate early development.

duration of each job. Cloud or cluster computing commonly in place for imaging-based analyses. The

AiryScan microscopy to study vision development

increasing growth in transcriptomics and genomics

by Ryan B. MacDonald and Elisabeth Kugler (University

data, however, promise

College London, UK)

services enable outsourcing of analyses, yet are not

expansion

cloud

computing, making it accessible for all applications in the future.

The retina, the light-sensitive tissue lining the back of our eyes, is constituted by various cell types that

Despite these hurdles, LSFM adds a valuable

are highly interconnected to form visual circuits.

dimension to live imaging using zebrafish. With

Müller glia (MG) are specialised glial cells that are

ISSUE 65 MARCH 2022

Figure 2.2. Multi-colour image acquisition, showing MG (green-blue) and retina nuclei (gray) in a 3-day old fish (scale bar 10µm).

responsible for supporting healthy retinal function

uses Zeiss AiryScan fluorescence microscopy, which

throughout life. To perform this job effectively, MG

enables image acquisition with 120nm lateral and

have a complex cell shape with apicobasal polarity

350nm axial resolution, to visualise glial cells in the

and defined subcellular regions that facilitate close

developing and ageing zebrafish retina.

associations with neighbouring cells and allows them

However, this state-of-the-art approach not only

to fulfil their specific supportive functions [18]. For

allows the visualisation of MGs, but also other

example, these MG subregions enable close contact

retinal components such as cell nuclei (Fig. 2.2;

with photoreceptor neurons, containment of the

average diameter 10 micrometers) or neurons in

cell nucleus, interaction with other neurons, and

parallel. With this, we can start to unravel how glia

ensheathment of blood vessels [19].

cells relate to visual function and the importance

Nevertheless, while MG shape is critical for their

of their interactions with other retinal components

function, it remains challenging to fully visualise and

(Fig. 2.3).

comprehend their complex morphology without

Taking this further from observational to functional

high-quality 3D visualisation (Fig. 2.1). Thus, our lab

imaging, our lab also uses approaches to visualise

Figure 2.3. 3D depth-coded image of neurons in the eye, called retinal ganglion cells, in a 2-day old fish (scale bar 10µm).

neuronal calcium dynamics to unravel the temporal

acquisition or post-processing. (b) When comparing

sequences of events in the developing retina around

Airyscan microscopy to other techniques, such

neuronal function and glial morphogenesis. Similarly,

as LSFM, the acquisition rate is slower, which can

using cell- or tissue-specific manipulations enables

make it tempting to change acquisition settings to

us to understand the role of different retinal cells in

increase speed. However, this comes at the cost of

the developing visual circuit.

sensitivity and resolution, making longer acquisition

Importantly though, like every microscopy technique,

durations inevitable for the highest quality data.

also Airyscan microscopy comes with its challenges.

Together, Airyscan microscopy can acquire data with

From an image acquisition perspective, we found

semi super-resolution, allowing the visualisation of

particularly two aspects needing consideration

small-scale cellular features that might be otherwise

across fields and experimentation: (a) Confocal and

overlooked and the understanding of the role of glia

Airyscan microscopy suffer from substantial z-axis

in retinal development and disease.

signal decay, which can be partially corrected during

ISSUE 65 MARCH 2022

Imaging and data analysis of brain function by Nathalie Jurisch-Yaksi and Emre Yaksi (Norwegian University of Science and Technology, NO)

behaviour [20], [21], as well as the function of nonneuronal cells (glia [22], ependyma, Olstad [24] and D Gama [28]). To do this, we employ a combination of two-photon, confocal, LSFM, and epifluorescence microscopy in larval and adult zebrafish. Here,

Understanding the function of brain circuits in

we discuss the advantages and disadvantages of

health and disease is a major challenge.This is mainly

these methods, and propose strategies on how

due to the immense complexity of the brain that

to combine them to study brain development and

is emerging from connections between millions of

function.

individual neurons and glial cells. Furthermore, this complex network further interacts with the rest of the body (e.g. cerebrospinal fluid, metabolites, hormones) and environmental signals, making a holistic study of the brain very challenging. With its small and transparent brain, zebrafish provide a complementary alternative to mammals for studying the entire nervous system in living animals.

Mostly we utilise two-photon microscopy for recording neuron and glia functionglia function (Fig 3.1) [20]-[22]. Two-photon microscopy allows excellent optical sectioning and depth penetration up to several hundred micrometres in awake behaving (free and head-restrained) animals, noninvasively in both larval and juvenile zebrafish. Yet, most two-photon microscopes rely on scanning

Our labs, individually and collaboratively, study

lasers, which limits the temporal resolution and

the physiology of neurons, glia and the ventricular

signal-to-noise ratio. To deal with such limitations,

systems in brain development and function.

many labs are now using LSFM (explained above)

Specifically, we study the role of internal brain states

to visualise brain function at several hundred

in sensory computations and regulating animal

frames per second (FPS). In fact, LSFMs were used

Figure 3.1.Two-photon microscopy is commonly used in combination with electrophysiological recordings. Here is a demonstration of glass electrodes that are used for stimulating and recording neural membrane potentials, while performing calcium imaging of neural activity (Photo credit: Anna Maria Ostenrath).

Figure 3.2.Tracking of the solitary motile cilia of five ependymal cells based on light sheet recordings in two-day-old animals.Time is colour-coded. (Olstad [24]).

to image the activity of all neurons and glia, across

separation

the entire vertebrate brain of larval zebrafish [23].

confocal microscopy is our method of choice when

This was a major achievement for neuroscience!

collecting anatomical or histological images from

In our labs, we use LSFM up to 950 FPS to image

brain samples with multiple colours. Moreover,

very fast biophysical processes such as the beating

excellent spectral separation is an advantage when

of cilia [24], [25] (Fig. 3.2), lining the brain ventricles

used for simultaneous functional imaging of multiple

or the nose. It is important to note that LSFM

cell types in the brain (e.g. neurons and glia) that

requires intricate sample preparation, which is

express indicators of activity in different colours.

easier to employ in smaller transparent larval fish. Moreover, most LSFMs rely on visible light, which has inadequate tissue penetration and can be problematic while imaging thick samples. Finally, visible light exposure is an additional concern when studying how it impacts on animals’ behavioural states, when studying how brain computations lead to behaviour as the light itself impacts the behaviour. Several labs now employ two-photon light-sheet microscopes, which give better optical sectioning and deeper tissue penetration than visible light and are not detectable by animals, therefore not affecting their behaviour.

different

fluorophores. Hence

However, advanced microscopes might not be available for most scientists in the world. Here, epifluorescence microscopes, some of which can be built for a few hundred dollars, can be very effective to study brain function. Epifluorescence microscopes cannot do optical sectioning of the tissue and present substantial light scattering some of these can be dealt with, using a few tricks. For example, we employ data analysis tools such as principal component- or independent component analysis to separate the dynamic neural signals coming from different locations in the sample. In brain regions with clear functional topography (e.g.

A major advantage of confocal microscopes is the

mice/fish/fly olfactory bulb, rodent barrel cortex)

range of lasers and filters coming with them, and

this approach has been successfully used to collect

their broad availability in core facilities (Fig. 3.3),

excellent quality neural activity data [26], [27]. We

which often means better excitation and spectral

also employ fluorophores with limited expression

Figure 3.3. Confocal stack of cilia and axons in a one month old zebrafish brain, with depth encoding. Staining done with an antibody against glutamylated tubulin [28].

ISSUE 65 MARCH 2022

Figure 3.4. Output of a 2p volumetric calcium imaging experiment done in four-day-old zebrafish with two light stimulations. It shows the complexity of the dataset and possible analyses allowing to recover the calcium dynamics in individual cells, similarities in responses across cell populations and their location within the brain.

to neuronal nuclei, or genetically defined cell types,

Similar to the ever more scientific questions

which helps greatly for identifying individual neurons

and techniques, optics and microscopy are ever-

or cell types despite the scattered light. Hence, we

changing fields. This is highlighted by the debut of

recommend scientists to consider available and

concepts such as tissue clearing and its combination

relatively cheap epifluorescence microscopes, which

with LSFM to push image quality boundaries even

often can be sufficiently good to answer many

further [29].

questions.

Current and future work also aim to advance the

It is important to note that functional brain imaging

frontiers of in vivo microscopy in larvae and adults

relies on fluorescent calcium indicators (Fig. 3.4),

as well as expand the library of functional imaging

which are well established, yet they are indirect and

tools.

slow reporters of neural activity. Moreover, calcium is

However, with evermore dimensions and data,

an ion that serves multiple functions in neurons and

one monumental challenge in research is how to

can be differentially released by different organelles

effectively handle, share, and analyse microscopy

independent of neural activity (i.e. ER, mitochondria).

data.

In fact, the source and function of glial calcium signals are still not fully understood, which complicates glial calcium imaging further. Hence, it is important to keep this in mind, when interpreting neuronal

The size and complexity of data sets require better computational frameworks, standardised software, and dedicated training of scientists. Many labs write their own software packages to

and glial calcium signals. Certainly, novel voltage

collect, store, analyse, and interpret results. It is

indicators will contribute to a better interpretation

not that uncommon for an hour-long experiment

of neural signals. Glial calcium imaging still needs

to generate hundreds of Gb of data, which then

further studies that will help us better interpret the

requires multiple steps of processing before

functional relevance of these signals.

scientists can assess results. Luckily several teams

Future Directions

generate open-access tools (e.g. FIJI, OASIS, CalmAN) that facilitate analysis of large data sets.

The examples discussed demonstrate not only

Yet, long-term visualisation and interpretation of

the breadth of fundamental questions studied in

large datasets remains a considerable challenge.

zebrafish but also the range of techniques required

One approach to reducing data duplication and

to do so.

supporting reproducibility is to openly share

microscopy data and analysis approaches, based on

resolution,” Nat Meth, vol. 12, no. 12, pp. 1171–1178,

the FAIR principle, namely Findability, Accessibility,

Dec. 2015, doi: 10.1038/nmeth.3632.

Interoperability, and Reusability [30].

[5] B. Schmid et al., “High-speed panoramic light-

An increased focus on data sharing and analysis

sheet microscopy reveals global endodermal cell

can provide new data insights and the discovery

dynamics,” Nat Commun, vol. 4, p. 2207, 2013, doi:

of important features that we might not know

10.1038/ncomms3207.

yet. What is clear however is that the increased digitisation and standardisation of microscopy data creates new opportunities towards the integration of different imaging modalities and the creation of multidimensional in silico datasets. Building on these, image-based computational “avatars”

[6] P. J. Keller, A. D. Schmidt, J. Wittbrodt, and E. H. K. Stelzer, “Reconstruction of Zebrafish Early Embryonic Development by Scanned Light Sheet Microscopy,” Science, vol. 322, no. 5904, pp. 1065– 1069, Nov. 2008, doi: 10.1126/science.1162493.

could be built to simulate and further understand

[7] A. Felker et al., “Continuous addition of

biological phenomena.

progenitors forms the cardiac ventricle in zebrafish,” Nat Commun, vol. 9, no. 1, p. 2001, May 2018, doi:

Conclusion

10.1038/s41467-018-04402-6.

We here showcased a wide range of techniques

[8] Y. Wan, Z. Wei, L. L. Looger, M. Koyama,

used in zebrafish to study - and answer - fundamental biological processes. What becomes clear is that one approach, technique, or model organism cannot provide all answers, but that combinations and multidisciplinary approaches are what spearhead scientific breakthroughs.

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[26] L. M. Franco, Z. Okray, G. A. Linneweber, B. A.

doi: 10.1017/S1431927617001817.

Hassan, and E. Yaksi, “Reduced Lateral Inhibition

[18] R. B. MacDonald, M. Charlton-Perkins, and W. A. Harris, “Mechanisms of Müller glial cell morphogenesis,” Curr. Opin. Neurobiol., vol. 47, pp. 31–37, 2017, doi: 10.1016/j.conb.2017.08.005. [19] E. C. Kugler, J. Greenwood, and R. B. MacDonald, “The ‘Neuro-Glial-Vascular’ Unit: The Role of Glia in Neurovascular Unit Formation and Dysfunction,” Frontiers in Cell and Developmental Biology, vol. 9, p. 2641, 2021, doi: 10.3389/fcell.2021.732820. [20] S. K. Jetti, N. Vendrell-Llopis, and E. Yaksi, “Spontaneous

activity

governs

olfactory

representations in spatially organized habenular

Impairs Olfactory Computations and Behaviors in a Drosophila Model of Fragile X Syndrome,” Curr Biol, vol. 27, no. 8, pp. 1111–1123, Apr. 2017, doi: 10.1016/j.cub.2017.02.065. [27] J. F. Christiaens et al., “The fungal aroma gene ATF1 promotes dispersal of yeast cells through insect vectors,” Cell Rep, vol. 9, no. 2, pp. 425–432, Oct. 2014, doi: 10.1016/j.celrep.2014.09.009. [28] P. P. D’Gama et al., “Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain,” Cell Reports, vol. 37, no. 1, Oct. 2021, doi: 10.1016/j.celrep.2021.109775.

microcircuits,” Curr Biol, vol. 24, no. 4, pp. 434–439,

[29] T. Chakraborty et al., “Light-sheet microscopy

Feb. 2014, doi: 10.1016/j.cub.2014.01.015.

of cleared tissues with isotropic, subcellular

[21] E. M. Bartoszek et al., “Ongoing habenular activity is driven by forebrain networks and

resolution,” Nat Methods, vol. 16, no. 11, Art. no. 11, Nov. 2019, doi: 10.1038/s41592-019-0615-4.

modulated by olfactory stimuli,” Curr Biol, pp.

[30] M. D. Wilkinson et al., “The FAIR Guiding

S0960-9822(21)01125–8, Aug. 2021, doi: 10.1016/j.

Principles for scientific data management and

cub.2021.08.021.

stewardship,” Scientific Data, vol. 3, no. 1, Art. no. 1,

[22] C. Diaz Verdugo et al.,“Glia-neuron interactions

Mar. 2016, doi: 10.1038/sdata.2016.18.

The authors Nathalie Jurisch-Yaksi, PhD @NathalieJuYa https://www.ntnu.edu/ikom/

Ryan B. MacDonald, PhD @MacDonald_Lab http://zebrafishucl.org/ macdonald-lab

cilia#/view/about https://

Ryan is a Biotechnology and

yaksilab.com/

Biological Sciences Research

(Norwegian University of

Council (BBSRC) David

Science and Technology, NO) Nathalie is a Group Leader at the Department of Clinical and Molecular Medicine at the Norwegian University of Science and Technology (NTNU). She is interested in non-neuronal mechanisms involved in brain development and physiology, with a main focus on cilia, glia, and cerebrospinal fluid biology.

Phillips Fellow and Lecturer at the Institute of Ophthalmology at University College London. He is interested in the cellular and molecular mechanisms regulating glial morphogenesis in the developing and ageing retina. Christian Mosimann, PhD @chrmosimann

Agnese Kocere, MSc @AKocere

Christian holds the Helen and

www.mosimannlab.org

Arthur E. Johnson Chair for

Agnese is a PhD student in the lab of Christian Mosimann at CU Anschutz and the Molecular Life Sciences Program of the University of Zurich, Switzerland. She is interested in the intersection of early embryo development and congenital disease and applies light sheet imaging in her research using zebrafish. Elisabeth Kugler, PhD @KuglerElisabeth www.ElisabethKugler.com Elisabeth is a Research Fellow at the University College London, producing biomedical image analysis approaches to quantify and model 3D glia morphology in the zebrafish retina. She is passionate about microscopy, science communication, and art; thus, sciArt plays an important role in her life.

www.mosimannlab.org

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the Cardiac Research Director and is Associate Professor of Pediatrics at the University of Colorado School of Medicine, Anschutz Medical Campus. He has a long-term interest in mechanisms of cell fate determination in development, disease, and evolution, a research direction he pursues with his lab using zebrafish as principal model. Emre Yaksi, PhD @yaksi_emre https://yaksilab.com/ (Norwegian University of Science and Technology, NO) Emre is a professor at the Kavli Institute for Systems Neuroscience at NTNU. He is interested in understanding how sensory information interacts with the internal dynamics of the brain, and how these processes modulate sensory computations and animal behavior.

Save the Date 25 -29 April 2022 Wiley Analytical Science Conference https://bit.ly/WAS-Conference-42022

https://analyticalscience.wiley.com

Wiley Analytical Science

Journal of

Microscopy The Journal of Microscopy publishes top quality research articles, review articles and Hot Topic papers covering all aspects of microscopy and analysis. This includes cutting-edge technology and innovative applications in physics, chemistry, material and biological sciences.

You can read the latest Early View papers online at www.journalofmicroscopy.org They include: Surface-enhanced Raman spectroscopy and ultrastructural analysis of penicillinproducing Penicillium rubens strains Carolina Campos, Luis Alberto Moreno Ruiz, Rogelio Fragoso-Soriano, Roberto Y. Sato-Berrú, Elizabeth Hernández-Pérez, Francisco J. Fernández Raman spectroscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques can perform chemical analyses and acquire high-resolution images of cell samples. For this reason, in this study, semi-thin sections of a single Penicillium rubens cell were analysed by Raman enhanced surface spectroscopy.The spectra showed peaks corresponding to the macromolecules that make up the cellular components. In addition, the various organelles were analysed by TEM and AFM to observe the cellular nanostructures. With the use of these techniques, it is possible to identify molecules in semi-thin sections, which provides a wide potential for biomedical applications and for the analysis of cell dynamics. The observation of the most detailed possible structure of cells is used as a starting point in numerous studies to identify and localise some biochemical processes. Given that the function of eukaryotic cells depends on the location, shape, structure, and function of the subcellular organelles

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(and on the interaction between them), the sum of the data obtained allows a complete analysis of what happens in the cell.This article addresses, from a multidisciplinary point of view, what happens in a single cell of a filamentous fungus (Penicillium rubens) while it is in a physiological moment (secondary metabolism) that allows the biosynthesis of an antibiotic (penicillin). For this purpose, different types of microscopies were used (TEM:Transmission Electron Microscopy, and AFM: Atomic Force Microscopy, which allow visualising small details in the cell) and a spectroscopy method (Raman, which allows detecting certain characteristics of the macromolecules and some stretching bonds). Regarding the results, during the synthesis of penicillin, the antibiotic-producing Penicillium rubens cells showed significant changes compared to the non-producing cells: the cell wall is observed to be significantly thickened in the production phase, organelles such as peroxisomes grow in number and size since it is known that the final route of metabolite synthesis takes place in them. When penicillin is released from peroxisomes, they must be degraded to release the load from the cell; this is done by vacuoles, which are active and engulf peroxisomes. The newly synthesised penicillin is found within secretory vesicles that travel towards the cell membrane and both membranes fuse creating ripples. On the other hand, and given that a

single cell is being studied, it is essential to increase the signal to detect biomolecules employing the Raman-SERS technique, using a silver substrate to obtain the increased signal.

Real-time monitoring of carbonation of hardened cement pastes using Raman microscopy Kai Zhang, Marcus Yio, Hong Wong, Nick Buenfeld This study investigated the feasibility of Raman microscopy for monitoring early surface carbonation of hardened cement pastes in real time for up to seven days. Samples were exposed to natural carbonation (440 ppm CO2) and accelerated carbonation (4% CO2), and the evolution of calcium carbonate (CaCO3) polymorphs, portlandite, ettringite, C-S-H gel and unreacted cement particles was followed. Results showed that calcite is the main polymorph formed under both natural and accelerated carbonation. Under accelerated carbonation, the formation of calcite on the sample surface completed within

one day whereas under natural carbonation, the formation of calcite is expected to continue beyond seven days. The contents of portlandite and ettringite decreased rapidly under accelerated carbonation but much more gradually under natural carbonation. However, calcium silicate minerals in unreacted cement particles remained unchanged throughout the carbonation processes. Overall, this study demonstrated that Raman microscopy is a valuable tool for non-destructive real-time imaging of surface carbonation in cement-based materials.

High-accuracy, direct aberration determination using self-attentionarmed deep convolutional neural networks Yangyundou Wang, Hao Wang, Yiming Li, Chuanfei Hu, Hui Yang, Min Gu Optical microscopes have long been essential for many scientific disciplines. However, the resolution and contrast of such microscopic images are dramatically affected by aberrations. In this study,

compacted with adaptive optics, we propose a machine learning technique, called the ‘phaseretrieval deep convolutional neural networks (PRDCNNs)’. This aberration determination architecture is direct and exhibits high accuracy and certain generalisation ability. Notably, its performance surpasses those of similar, existing methods, with fewer fluctuations and greater robustness against noise. We anticipate future application of the proposed PRDCNNs to super-resolution microscopes.

Assessment of the self-healing capacity of cementitious materials through active thin sections Emanuele Rossi, Claudia Romero Rodriguez, Henk Jonkers, Oğuzhan Çopuroğlu Since self-healing of cementitious materials can theoretically improve the service-life of concrete structures, it has gathered significant attention from both researchers and industry during the last two decades. Many researchers have proposed different methods to assess and quantify the self-healing capacity (i.e. the ability of cementitious materials to heal cracks) that is generated in concrete autogenously as well as autonomously. Even though many methodologies can be found in the literature, a way to accurately quantify the healing products produced by any selfhealing mechanism has not yet been achieved. In this study, a methodology is proposed to observe and to quantify in-time formation of healing products based on active thin sections. Thin sections of Portland cement paste have been prepared with no epoxy impregnation to facilitate reactions between the cement matrix and the surrounding environment. Artificial cracks (260 μm wide) were induced at 28 days of age and the crystal growth was continuously monitored up to 28 days of self-healing. Through image analysis of the micrographs, it was calculated that the autogenous self-healing capacity of paste (triggered by portlandite carbonation in uncontrolled indoor conditions) was around 55%

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after 28 days of self-healing. Healing products were further characterised through Environmental Scanning Electron Microscope analysis. Based on the results obtained in this study, the proposed methodology seems to be promising to compare the self-healing mechanisms triggered by different healing agents.

Implications of gnomonic distortion on electron backscatter diffraction and transmission Kikuchi diffraction Chris M. Fancher, Matthew J. Burch, Srikanth Patala, Elizabeth C. Dickey The effect of gnomonic distortion on orientation indexing of electron backscatter diffraction patterns is explored through simulation of electron diffraction patterns for sample-to-detector geometries associated with transmission Kikuchi diffraction (TKD) and electron backscatter diffraction (EBSD). Simulated data were analysed by computing a

similarity index for both Hough transformed data and simulated patterns to determine the sensitivity of each method for detecting subtle differences in the effect of gnomonic distortions on electron diffraction patterns. These results indicate that the increased gnomonic distortions in electron diffraction patterns for a TKD geometry enhance the sensitivity for detecting subtle differences in interband angles. Additionally, the utilisation of a Hough transform-based indexing approach further enhances the sensitivity.

What is the structure of our infrastructure? A review of UK light microscopy facilities Georgina Fletcher, Kurt I. Anderson Core Facilities and Technology Platforms are increasingly important components of the science research landscape. However, data on facility operations and staff careers are lacking to inform their development. Here we have surveyed 114 people working in 46 light microscopy (LM) facilities within the United Kingdom. Our survey explores issues around career progression, facility operations and funding. The data show that facilities are substantial repositories of equipment and knowledge which adapt to meet the needs of their local environments. Our report highlights the challenges faced by facility managers, institutions and funders in evaluating facility performance and devising strategies to maximise the return on research funding investment.

Submit to the Journal of Microscopy 1. 2. 3. 4. 5. 6.

No submissions fees No page or colour charges No page limit Simple online submission Helpful, friendly editorial team Average time from submission to first decision is less than 50 days 7. High readership figures 8. Online tracking system – authors can easily check the status of an article in production and receive emails at key stages 9. Rapid publication with Early View papers published online in advance of print, significantly shortening time from acceptance to publication 10. Free electronic offprints

Journal of Microscopy App Available for iPhone and Android

Search for Journal of Microscopy on the App Store or Google play and access your personal or institutional subscription wherever you are, whenever you want.

Submit online at https://mc.manuscriptcentral.com/jmi View the Guidelines for Authors and full submission details online at:

www.journalofmicroscopy.org 35

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Darwin’s microscope fetches almost £600,000 at auction Instrument is believed to have been used for naturalist’s influential work on ‘zoophytes’

n antique microscope belonging to legendary British naturalist Charles Darwin made international headlines in December, when it was sold at auction for an eye-watering £598,500. The instrument, designed by English microscope-maker Charles Gould for the Cary Company around 1825, had been passed down through the Darwin family for almost 200 years before going under the hammer at Christie’s in London. Vastly exceeding its top estimate of around £350,000, it is one of just six existing microscopes known to have been associated with Darwin, and the only one ever to have been put on the market. James Hyslop, Head of Department, Scientific Instruments, Globes & Natural History at Christie’s said: “The microscope was acquired for the successful collector through Gurr Johns advisory, who hopes many more Darwin enthusiasts will be able to enjoy the microscope on display in a public institution in the future.”

At the time of the microscope’s manufacture, Darwin was studying ‘zoophytes’ such as corals, sea anemone and sponges from the Firth of Forth – research famously carried out at the expense of his medical studies, and which would culminate in his first scientific paper presented to the University of Edinburgh’s Plinian Society in 1827. It is highly likely that Darwin used the instrument for these studies, since the only other he is known to have owned at this time would not have been suited to the task.The remaining four microscopes known to have been used by Darwin were acquired later (two in 1831, and a further two around 1847 - 1848) While Darwin is believed to have used one of these later microscopes during the iconic HMS Beagle Figures 1&2. Darwin's microscope photographed prior to its sale. Christie's Images Ltd. 2021 Figure 2.

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voyage (an instrument currently on display at his former home, Down House), Darwin’s work on

3(b)

zoophytes was also hugely significant, contributing to zoologist Robert Edmond Grant’s radical reinterpretation of the animal kingdom, which in turn heavily influenced Darwin’s thinking during, and immediately after the second voyage of the Beagle from 1831-1836. Darwin’s seminal publication ‘On the Origin of Species’ was eventually released in 1859. RMS History Committee Chair Dr John Hutchison, Hon FRMS notes: “For anyone interested in the history of science and microscopy, this is quite a story. The sale price is absolutely amazing, but when

3(c)

you consider the microscope’s provenance and the colossal importance of Charles Darwin in science history – or history full-stop, for that matter - it is perhaps understandable that a wealthy collector would be willing to part with that sort of money. In terms of the microscope itself, the RMS has three 3(a)

similar models in its collection at Oxford’s Science History Museum (Figure 3 a-c) – though sadly we cannot claim these particular instruments had anything to do with Charles Darwin!” In addition to the ‘Beagle’ microscope on display at Down House, the property is also home to a small botanical microscope, and another instrument by the Cary Company. The other two existing microscopes associated with Darwin are a large Smith & Beck compound microscope (held in the collections of the Whipple Museum in Cambridge) and a prototype of a modified aquatic microscope developed by Darwin himself. This design was put into wider production by Smith & Beck in the 1840s. The inventive term ‘Darwinania’ has been coined for items of memorabilia associated with the celebrated naturalist. Figure 3 (a, b and c):The RMS has three similar models in its collection.

Owen Morton

New Member Welcome The Royal Microscopical Society would like to welcome our new members who have joined us in the last three months.We hope they enjoy a long and rewarding membership with the RMS. Dr Ana Stojiljkovic Ms Nandini Aggarwal Mr Daohe Yuan Mr Alexis Gkantiragas Miss Francesca Willcocks

Dr Kathy Newell Dr Sargon Gross-Thebing Dr Nicholas Anthony Professor Ruben Cauchi Dr Shamily PB

Mr Jack Taylor Miss Bijal Amin Dr Davide Randazzo Miss Nadine Field Dr Christian Matek Dr Andrew Chandler-Grevatt

If you know of anyone who might be interested in becoming a member of the Royal Microscopical Society and you would like us to contact them, please send their details to our Membership Administrator, Debbie Hunt – membership@rms.org.uk. Application forms are available to download at www.rms.org.uk/membership. Don't forget you can now log into the RMS website and check your membership status, renew and download receipts. If you have never logged into the RMS website you can register your details to gain access, please remember to use the email address that is linked to your membership. If you have any queries or questions about your membership please contact Debbie Hunt debbie@rms.org.uk

Member Profiles Name

Samson Oyindamola John Tell Us About You? Oyindamola Samson is a Graduate Research Assistant. He obtained his certificate of Achievement in COVID-19: Tackling the Novel Coronavirus Course at London School Of Hygiene & Tropical Medicine And UK Public Health Rapid Support Team In 2020. He is currently based at the Antimicrobial and Natural Products Research Laboratory as a Graduate Research Assistant. Oyindamola Samson’s research focus is on Microbiology and Biotechnology. Why did you become a member of the RMS? Societies are a kind of enclosure which makes research ideas and innovation powerful and effective to change the world. Provide training to young engineers and scientists by conducting workshops and certified online courses.

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Provide a forum for presenting our work (magazines, conferences and journals). SPIE, ASME, SMST etc. used to conduct yearly conferences and have their credible international journals. How do you feel being an RMS member benefits you? Societies like RMS are a great way to get advice, access dedicated support and training, or to meet and learn from role models in your field. Many place a particular focus on developing graduate students or early-career researchers. I will be able to build confidence through finding and sharing with other people like myself via the platform of RMS.

Name

Siraj Khan Tell Us About You? I'm working as a PhD student in the department of Botany Abdulwali Khan University Mardan, Pakistan and recently working as a Manager in Qarshi Herb Research Center in Qarshi Industry (pvt.) Ltd.

Name

Rahul Mehta Tell Us About You? In Max Planck Research laboratory situated at Malopolska Center of Biotechnology, Krakow, Poland. I am pursuing my doctoral study under Dr Sebastian Glatt’s supervision and my current work is focused on the structural studies of multiple, folded RNAs using single-particle cryo electron microscopy. Why did you become a member of the RMS? To engage with the microscopy community and keep myself up to date with latest developments

Name

Alan Neale Tell Us About You? 25 years in microscopy, evaluating materials for industry. I began at the British Ceramic Research Association, looking at ceramic and high temperature refractory microstructures. More recently I completed studies for the consumer healthcare sector, assessing the effect of toothpaste formulation on enamel mineral density; and for the medical devices sector,

Why did you become a member of the RMS? I have recently been working on light and scanning electron microscopes, linked with plants, including pollen study, anatomy etc, and now I want to share and gain some extra knowledge. How do you feel being an RMS member benefits you? I'm feeling proud because I'm a member of such an old society working for the betterment of research.

in the field along with getting help through various training modules run by RMS from time to time. How do you feel being an RMS member benefits you? By staying up to date with various activities in the field, we grow a better understanding of the field itself, its trends and emphasis.

evaluating coating integrity of vascular stents and wear of orthopaedic implants. Currently running the microscopy section at Lucideon. Why did you become a member of the RMS? To expand my understanding of what is a rich and diverse subject. It is easy to get caught up in your own narrow field of interest; it’s good to see developments in the wider community. How do you feel being an RMS member benefits you? infocus is always a fascinating read. Articles covering the history of microscopy sit alongside articles on its future and some of the imaging features blur the line between science and art.

Name

Avnish Singh Pal Tell Us About You? I am a Research Scholar at the Indian Institute of Technology (BHU), Varanasi, working in the field of transmission electron microscopy of advanced materials for spintronics and energy storage devices. Why did you become a member of the RMS? With the help of RMS, I want to update myself regarding the latest technical updates and job

Name

Michael Jalil Khayat Tell Us About You? I am a PhD student pursuing a degree in Mining and Materials Engineering at McGill University. I received my BS in Ceramic Engineering and minors in Biomedical Engineering and French in May 2021. My project revolves around integrating sol-gel borate-based bioactive glass into a dense collagen matrix and investigating their potential in regeneration of mineralised and nonmineralised tissues.

Name

Brynn Dachtler Tell Us About You? I am an Optical Test Engineer at Covesion. I have previously been a Test and Characterisation Engineer at CoolLED, and also part of a PhD programme at the University of Southampton to study Tribology. The first Christmas card I received from the RMS had a Transition Metal Dichalcogenide on the cover, MoS2, which was very interesting to me as I was studying it at the time. Why did you become a member of the RMS? During my time at the University of Southampton I did a lot of work with AFM and

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opportunities in the field of Transmission Electron Microscopy. How do you feel being an RMS member benefits you? RMS is a platform that provides all the necessary help to a researcher such as technical updates, conference updates, job updates, etc.Above all, it gives us a platform to connect with the research community of this specific field.

Why did you become a member of the RMS? I became a member of RMS to broaden my network and immerse myself in a larger pool of cutting-edge and innovative peers. How do you feel being an RMS member benefits you? I feel that joining RMS provides access to a wealth of opportunities, connections, and collaborations that will challenge me and bring out my best work.

Raman Microscopy, and one of my office mates was a member and it seemed very interesting. Afterwards as I moved to CoolLED to work more with optical microscopy, I kept up the membership as it allows me to learn about lots of different news and applications of microscopy in the wider world. How do you feel being an RMS member benefits you? It gives me interesting stories from the world of microscopy outside my current experience zone. I feel proud to call myself an RMS member and it allows me opportunities to network further.

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AU T H O R I N T E RV I E W

Infectious

by Dr. John Tregoning Celebrating research accomplishments and history of the last hundred years in immunology, virology, microbiology, epidemiology in a book.

RMS Ambassador Niga Nawroly, of Agilent Technologies, puts the questions to Dr Tregoning, author of the recently published Infectious.

ohn is reader in respiratory infections at Imperial College London, working on the St. Mary’s campus, close to the labs where Alexander Fleming discovered penicillin. His book Infectious: Pathogens and how we fight them celebrates the history of science, diagnostics, alluding to the history of research techniques such as microscopy, imaging, other immunological techniques, and drug discovery. He describes some of the historic perspective on microscopy that led us to understand pathogens and how they infect us; particularly how van Leeuwenhoek’s research pulled the curtain back on the microbial world. NN: What is it that you do as a reader in immunology? JT: I lead a team of researchers. Currently there’s five PhD students in my lab and they are working on various projects we’re interested in. I also teach, and then writing the papers and the grants that come out of the work that my team does. NN: Are you also involved in conducting experiments yourself? JT: Whilst the techniques have moved on considerably since I was most active in

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the lab, I do still help sometimes, but I think probably we’ve got to the point where I’m more disruptive in the lab than helpful. NN: What is it you like about your job and why are you doing this? JT: I love the fact that I get to work with interesting, brilliant people from all around the world; getting to talk about science and collaborate on interesting projects. The second reason is answering the scientific questions. I like problem solving: I’m more interested in answering fundamental

questions rather than necessarily making new therapies. And thirdly, as was described by Hope Jahren, in her book Lab Girl, there is the occasional moment where you know something that nobody else in the world has discovered and you have a burst of excitement.

JT: I try (but often fail) not to be too emotionally attached to each individual experiment. If at any one time, one of my team's experiments isn't working, another one is working. So, I get to live through the exciting parts and not have to deal with the emotional baggage afterwards; up to a point.

NN: Can you describe a particularly memorable ‘wow’ moment in your career when you looked down a microscope?

NN: In answering your scientific questions, how are you using flow cytometry and imaging techniques?

JT: The first time that happened to me was when I was an undergrad in Cambridge. We were looking down a microscope at a Drosophila larva in which we induced a mutation. My supervisor at the time said, "you know you're the first person ever to see this", and that kind of thinking stuck with me. These days in science, in general, moments of success are few and far between, but they’re worth celebrating when they happen.

JT: To do this I’m mostly using the simplest technique possible to get you the best answer. We are using flow cytometry to look at cell phenotyping. Focusing on small panels that are answering a question rather than something big. Simple and robust is my preferred approach. As for imaging, we are looking at protein expression to answer questions related to adaptive immune responses to vaccines. We also use in vivo imaging to follow the expression of vaccine antigens: we have just published on how altering T cell migration around the body changes the expression of DNA vaccine

NN: What is driving you when experiments fail, especially when you have a brilliant idea on paper but fail to deliver the anticipated results?

encoded luciferase. (https://onlinelibrary. wiley.com/doi/10.1111/imm.13429) NN: What is your approach in standardising the techniques you use? JT: We are very lucky we have a very well supported flow cytometry facility at Marys. Radhika Patel, who runs the core facility helps us with panel design and all other cytometric challenges. We rely on her expertise in that area, because the technology is changing faster than the people in the lab. So, I see the importance of having a specialist to support me to do it properly. NN: Please tell us about your book. Who is it aimed at? JT: It is aimed at everyone from GCSE, upwards. There’s enough in there for specialists to read outside their fields, so if you’re an immunologist, there’s lots of microbiology. For Virologists, there’s immunology, epidemiology. It’s for anyone

who’s interested in infectious disease or would like to know more about what we’ve just been through including some COVID information. NN: You talk about the history of vaccines, the different vaccines against COVID, why are they so important? JT: Vaccines work like seatbelts, they’re preventative. Vaccines are very effective. Particularly they are cost effective; the tetanus vaccine is a dollar a shot, a treatment in ICU is going to be $10,000. NN: What is your experience with vaccine work? JT: My research is experimental (preclinical) vaccines. My first ever paper was on edible vaccines in plants. It was the first demonstration that plant vaccines could be protective against an infection. (https://academic.oup.com/nar/ article/31/4/1174/2375901)

Figure 1.Taken from the paper 'A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging' https://pubmed.ncbi.nlm.nih.gov/26091084/.

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Figure 2.Taken from the paper 'Airway T cells protect against RSV infection in the absence of antibody' https://www.nature.com/articles/mi201746.

From those early studies at the beginning of the 21st century, other groups and companies have now developed and demonstrated the efficacy of a plant-derived flu vaccine in phase III clinical trials. NN: How important do you feel vaccines have been in the response to the COVID-19 pandemic?

JT: Building on all the experience generated in the last century and the first 20 years of this one, we have seen a great leap forwards in vaccines in 2020-21. That there were vaccines ready for use within a year of a new viral pandemic starting speaks to the incredible progress we have made. Niga Nawroly

2 0 2 1 S U M M E R S T U D E N T S H I P R E P O RT

Localisation and distribution of the activityregulated cytoskeletonassociated protein (Arc) in the mammalian brain Student: Barbara Altenhuber Supervisor: Dr René Frank, University of Leeds Project location: University of Leeds During this internship, I learnt important techniques in sample preparation of brain tissue for microscopy and the use of widefield as well as confocal microscopes to visualise the anatomical distribution of proteins in the brain. Using these two different microscopes, I was able to get a glimpse of a fascinating hidden world. I enjoyed developing and re-assessing workflows to achieve the best visualisation of different brain regions. These workflows ranged from sample preparation to the settings of the microscope. Often minute changes in the workflow affected the experiment and, therefore, needed to be re-evaluated and, if necessary, adapted. Additionally, I learnt to process images taken on the cryogenic electron microscope (cryoEM). Participating in this internship has assured me in my

the hypothesised Arc capsids. After completing my

ambition to pursue a career in research. The use

project next year, I wish to stay in active research

of microscopes in this project has awakened my

and start a PhD.

interest in different microscopes and the physics as

well as engineering underlying them. I am grateful

Summary

to Professor Seth Grant, University of Edinburgh,

A host of endogenous retroviral sequences

for providing me with the ArcVenus mouse line that

‘colonise’ the human genome. These sequences

enabled these studies. I am looking to utilise the

have become integrated into the human genome,

knowledge and experience gained during this

hence the term endogenous retroviral sequences.

internship in the next stages of my career. I am

However, it is still unknown if these genes serve

fortunate to be staying with the same lab group

physiological functions in the human body. The

for my final year project and, thus, I will be trained

activity-regulated cytoskeleton-associated protein

in the use of the cryoEM to continue the hunt for

(Arc) belongs to these endogenous retroviral genes

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and is known to be involved in regulating structural

To address this aim, a new mouse strain containing

changes during synaptic plasticity, an important

an endogenous tag of Arc was used to visualise

process in learning and memory. Excitingly, Arc

the protein. Locating Arc has been challenging

self-assembles into virus-like capsids in vitro. These

because of the poor specificity of Arc antibodies

mRNA-containing capsids are hypothesised to

(Pastuzyn et al., 2018). To overcome this limitation,

be transferred across synapses, mediating a novel

an in-frame fusion of a yellow fluorescent protein

intercellular communication pathway in the brain

(YFP) tag to the Arc gene (ArcVenus) was used to

for which structural evidence is still needed in vivo.

identify the subcellular structures within which Arc

Despite the apparent importance of Arc in synaptic

resides (Fernández et al., 2017). Brains of ArcVenus

plasticity and the hypothesised viral pathway, the

and wildtype mice were frozen in optimal cutting

distinct location of Arc remains unknown. The

temperature (OCT) medium before sectioning into

location of Arc in the mammalian brain is vital to

14 μm thick slices on a cryostat and mounting onto

understanding the function of Arc in the mammalian

slides (Frank et al., 2016). Fluorescent markers were

brain and forms the foundation for the investigation

used to co-stain cell nuclei (DAPI) as well as pre-

of virus-like capsids involved in cognition. The

and postsynaptic compartments with synapsin 1

objective of this project was to determine the

and PSD95 antibodies, respectively. These samples

location of Arc in the mammalian brain.

were then imaged using fluorescence microscopy (widefield, confocal microscopy; Leeds Bio-Imaging Facilities) to establish the location of Arc in different brain regions as well as subcellular compartments. Initial widefield images of the ArcVenus mouse brain presented with low Arc signal that was only slightly higher than in control mice lacking the endogenous Venus tag.To verify that the Venus tag was expressed in the sample, a Western Blot of the ArcVenus tissue was done. This Western Blot showed that the YFP-tag was indeed attached to the Arc protein. Nevertheless, the protein could not be visualised, which we concluded could be because of two reasons: (1) fluorescence signal was too low because a step during the sample preparation affected the Venus-tag. (2) Physiological Arc levels might not be sufficiently high enough for visualisation. To eliminate the possibility of the fixative affecting the endogenous tag, the fixative was changed from methanol to paraformaldehyde. However,

Figure 1.Working on the confocal microscope at the Leeds Bio-Imaging Facility.

Report This project aimed to determine the localisation of Arc in mammalian brain regions as well as its subcellular distribution.

the samples fixed with methanol and PFA did not differ in the observed Venus tag signal. Therefore, to enhance detection of low levels of ArcVenus, a green fluorescent protein (GFP) antibody was used that recognises the homologous Venus tag. With the use of the GFP antibody, the Venus-tag signal could be enhanced, and Arc protein could

Figure 2. Sagittal image of the ArcVenus mouse under the widefield and confocal microscope. ArcVenus (GFP antibody), green. Cell Nuclei (DAPI), blue. A) Whole brain. Scale bar, 1 mm. B) Hippocampus. Scale bar, 1 mm. C) Cerebellum. Scale bar, 100 µm. D) Granular Layer of the dentate gyrus.The image was taken from optical sectioning. Scale bar, 20 µm. E) Dentate gyrus. Examples of dendrite-like extensions are marked with a white arrow. Scale bar, 100 µm.

be localised in the cortex (Fig. 2A), hippocampus (Fig. 2B), and cerebellum (Fig. 2C) and was absent

References Fernández, E., Collins, M. O., Frank, R. A. W., Zhu,

in wildtype control samples. Arc showed a high co-

F., Kopanitsa, M.V., Nithianantharajah, J., Lemprière,

localisation with DAPI, a marker for cell nuclei and,

S. A., Fricker, D., Elsegood, K. A., McLaughlin, C. L.,

therefore, was particularly abundant in the granular

Croning, M. D. R., McLean, C., Armstrong, J. D., Hill,

layer of the cortex and dentate gyrus, as well as the

W. D., Deary, I. J., Cencelli, G., Bagni, C., Fromer,

stratum pyramidale of the cornu ammonis regions

M., Purcell, S. M., Pocklington, A. J., Choudhary, J.

of the hippocampus, which are cell body-dense

S., Komiyama, N. H. & Grant, S. G. N. 2017. Arc

regions. Optical slicing through the sample showed

Requires PSD95 for Assembly into Postsynaptic

that Arc signal is contained within the cell nucleus

Complexes Involved with Neural Dysfunction and

(Fig. 2E). Arc signal also extended beyond the cell

Intelligence. Cell reports, 21, 679-691.

nucleus stained by DAPI, suggesting that Arc is also present in the perinuclear region. Furthermore, Arc signal could be found extending from the cell bodies resembling the shape of dendrites (Fig. 2D). Strong evidence for the distribution of Arc in pre- and postsynaptic markers could not be produced within

Frank, R. A. W., Komiyama, N. H., Ryan, T. J., Zhu, F., O’Dell, T. J. & Grant, S. G. N. 2016. NMDA receptors are selectively partitioned into complexes and supercomplexes during synapse maturation. Nature Communications, 7, 11264.

the scope of the internship. Nevertheless, the finding

Pastuzyn, E. D., Day, C. E., Kearns, R. B., Kyrke-Smith,

that Arc is abundant in the cortex, hippocampus and

M., Taibi, A.V., McCormick, J.,Yoder, N., Belnap, D.

cerebellum, is perinuclear as well as nuclear and is

M., Erlendsson, S., Morado, D. R., Briggs, J. A. G.,

present in dendrites is an important step towards

Feschotte, C. & Shepherd, J. D. 2018. The Neuronal

understanding the proposed virus-like pathway of

Gene Arc Encodes a Repurposed Retrotransposon

Arc.

Gag Protein that Mediates Intercellular RNA Transfer. Cell, 172, 275-288.e18.

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FIND OUT MORE!

2 0 2 1 S U M M E R S T U D E N T S H I P R E P O RT

Image processing and analysis methods for the detection of islets in Mesolens images of whole mouse pancreas specimens Student: Mollie Brown Supervisors: Professor Gail McConnell & Ms Katrina Wesencraft, University of Strathclyde Project location: Carried out remotely The aim of this project was to develop an image processing and analysis pipeline to identify and quantify islets in confocal fluorescence Mesolens images of whole mouse pancreas specimens. This project was carried out remotely and was entirely

The majority of the work was undertaken using FIJI

computational, using image datasets produced by Ms

[1], an image processing and analysis programme,

Katrina Wesencraft at the University of Strathclyde.

using various plugins. One of the key plugins used,

The majority of the work was undertaken using FIJI

CLAHE [2], is a tool that breaks the image into

[1], an image processing and analysis programme,

multiple small sections, and increases the contrast

using various plugins. One of the key plugins used,

within each, resulting in a locally contrasted image as

CLAHE [2], is a tool that breaks the image into

opposed to carrying out the procedure on the whole

multiple small sections, and increases the contrast

image, and therefore overly contrast-adjusting some

within each, resulting in a locally contrasted image as

regions. Alternative plugins such as Trackmate [3], a

opposed to carrying out the procedure on the whole

plugin designed for particle detection and tracking,

image, and therefore overly contrast-adjusting some

had some success for islet detection as this sought

regions. Alternative plugins such as Trackmate [3], a

out intensely fluorescent areas of a circular shape.

plugin designed for particle detection and tracking,

The in-built FIJI functions could also be used for islet

had some success for islet detection as this sought

detection, as once an intensity threshold was applied

out intensely fluorescent areas of a circular shape.

to the data, it was possible to use the “Analyse

The in-built FIJI functions could also be used for islet

particles” function to detect circular shapes within

detection, as once an intensity threshold was applied

images, based upon an approximate area.

to the data, it was possible to use the “Analyse particles” function to detect circular shapes within images, based upon an approximate area.

Findings Compared to the raw datasets, the images processed

This project was carried out remotely and was entirely

using CLAHE provided clearer discrimination

computational, using image datasets produced by Ms

between islets and the rest of the pancreas tissue.

Katrina Wesencraft at the University of Strathclyde.

Whilst this was an improvement and provided a

ISSUE 65 MARCH 2022

Figure 1. A comparison between an original image (A), and the altered image, after using CLAHE, and enhanced contrast (B).

clearer, brighter set of images, there was still the

based on their non-circular shape. Unfortunately, the

persistent issue where the islets within the pancreas,

restrictions in size range required to eliminate false

which are visible as bright spots, were not always

detections also meant that many smaller islets were

clear initially and each image had a different level of

being missed. Whilst these methods both had some

brightness.This was combatted through enhancing the

level of success and provided an automated method

contrast within the dataset and using the histogram

of detection, they both relied on parameters being

of the stack, such that for all images, the brightest

set each time based on the individual dataset.

spot in each had the same grey value and no singular image would be exceptionally brighter or darker than the others within the stack. Once the parameters had been set, this process was able to run automatically. Fig. 1 demonstrates the difference between one of the original images and the same slice after editing.

On top of using automatic detection methods, I also hand annotated a selection of the images, following the appearance and development of islets throughout the pancreas, by circling each clearly visible spot with an editing tool. This acted as a comparison for the automatic detection methods, but it took significantly

Automatically detecting the islets proved to be more

longer to perform. Manual annotation took around

difficult as islets vary in size from 50 to 500 microns

1 hour, to analyse only 41 images, compared to the

and the differences in size and intensity meant that

almost instantaneous analysis of 178 images using the

not all could be identified. Additionally, many methods

‘Analyse particle’ function.

that I tried often picked up false positives, detecting spots where there were none. For example, the Trackmate plugin was able to highlight many islets throughout the images: by setting an approximate diameter range, it automatically detected any spots of that size. However, as shown in Fig.2, it captured a lot of false positives, where the image was brighter but where there were not islets. Another more successful method was using the FIJI ‘Analyse particle’ tool as discussed above and seen again in Fig. 2. This captured some areas of high intensity but was able to disregard them

Learning from participation in project Before undertaking this internship, despite having previously used coding languages such as MATLAB, to develop figures, and read image data, I had no strong experience in image processing or image analysis.This project has allowed me to not only develop these analysis skills, learning the basics of creating and using macros to analyse images, and becoming comfortable with using programmes such as ImageJ, but it has also allowed me to refine my own broader computational skills along with a general understanding of this area,

despite it not being an area covered within my course curriculum. I’ve discovered on a personal level that I enjoy the opportunity to work on a project, and to undertake work that requires a level of self-management, tackling a problem from start to finish, and the research and work required to do so. Whilst also gaining the opportunity to work collaboratively with my project supervisors, who’s expertise and advice was invaluable, and I can’t thank them enough for giving me the opportunity to work with them on this. I also thank the Royal Microscopical Society for funding this project so that I could gain this invaluable experience.

Impact on long-term goals Completing this project has sparked my keen interest in microscopy, and its applications, not only within physics (despite my background there), but within other areas of research. It has also encouraged me to start looking at career opportunities in this area. Undertaking this internship has given me an insight into some of the practicalities of a research career. I am interested in pursuing my newly discovered interest in microscopy, whether that be through continuous learning within employment after graduating, or in an ideal situation, I would love to undertake a PhD. A PhD would allow me to further develop my knowledge and expertise, whilst contributing to the exciting and diverse field of light microscopy. [1} Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. [2} Zuiderveld, K. (1994) Contrast limited adaptive histogram equalization. Graphics gems IV, pp. 474–485. Academic Press Professional, Inc, San Diego, CA, USA

Figure 2:The original image (A), the image analysed using Trackmate, with islets identified by red boundaries (B) and the results using ‘Analyse particles’ function, with islets highlighted in red (C).

ISSUE 65 MARCH 2022

[3} Tinevez, J.-Y., Perry, N., Schindelin, J., Hoopes, G. M., Reynolds, G. D., Laplantine, E., … Eliceiri, K. W. (2017). Trackmate: An open and extensible platform for singleparticle tracking. Methods, 115, 80–90. Molly Brown

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2 0 2 1 S U M M E R S T U D E N T S H I P R E P O RT

High throughput imaging and analysis for studying feeding and digestion in a marine invertebrate model Student: Elene Lominadze,Theoretical Physics BSc, University College London Supervisor: Dr Alex Corbett Project location: University of Exeter The “Multiscope” (also known as the Rapid Access Parallel microscope) system enables multiple fields of view to be relayed to the same detector providing applications in high-throughput imaging. The Multiscope is the product of a collaboration between the groups of Dr Alex Corbett (Exeter University) and Prof Gil Bub (McGill University) [1]. In this project the Multiscope has been implemented as a multi-well plate reader to analyse the movements of microscopic (~200μm) larvae of the marine worm Platynereis dumerilii. Platynereis is an excellent model for studying the

Multiscope to identify changes in feeding behaviour

regulation of feeding and digestion as it is easy to

by extracting parameters such as speed and

keep in the lab and the small size and transparency

trajectory of each larva. Imaging multiple fields of

of Platynereis larvae and juveniles mean that the

view in parallel improves reproducibility, which is a

entire digestive process can be imaged in live, freely

recognised issue in life sciences [5].

moving individuals [2]. A variety of neuropeptides

Method

can be used to modify appetite, food search behaviour and gut motility in Platynereis. The neuropeptide complement largely overlaps with that of other organisms [3], including mammals, allowing us to study the function and evolution of specific conserved neuropeptides in the regulation of animal digestive systems. Modulations in the behaviour of Platynereis were identified by analysing Multiscope images of the larvae in different feeding environments.

Throughout the project, videos of freely-moving larvae were generated using the Multiscope in the presence or absence of food. The Multiscope can image multiple spatially separated samples at high-resolution without movement of the sample or the imaging system, allowing us to image nine wells occupied by the larvae in parallel. Initially, ~18 larvae were placed in each well filled with sea water and covered with a cover slip to avoid a meniscus. The larvae were imaged, and later fed with algae to

Aim

be imaged again, so as to compare the difference

The aim of the project was to use bright field

in larval behaviour in the presence and absence of

images of the Platynereis larvae captured with the

food. 900 images were taken before, and 900 after

ISSUE 65 MARCH 2022

Figure 1. An illustration of the Multiscope (left) used to provide high throughput screening by imaging multiple wells in parallel. Multiscope illumination and frame capture are controlled using a Raspberry Pi at the base of the Multiscope.

feeding (100 images per well) at a rate of 1 frame

increase the field of view to ~80% of the well area.

per second (fps).

The next experiments focused on validating the

The nine fields of view were first stitched together into a video file using a custom Python script. Automated visual tracking software, ‘TRex’ [4], was then used to further process the videos to extract features of interest, such as speeds, trajectories and visual fields. As an alternative to TRex, the ‘OpenCv’ Python module was used for analysis as it contains all of the main image and video processing functionality. In the comparison, TRex was favoured

method of using tracking to measure changes in larvae behaviour by looking at changes before and after the addition of food (algae). Following data analysis, it could be seen that the speeds of the larvae increased post-feeding as expected (Figure 4), validating the approach. The next step would be to monitor the influence of different neuropeptides on behaviour, but time constraints meant that these experiments were beyond the scope of this study.

due to its additional features such as estimating

Analysing the obtained data using the tracking

the visual fields of each larva based on midline

software turned out to be more challenging than

orientation.

anticipated – initially, the larvae frequently moved in

Results and Discussion

and out of the field of view, causing problems with

Initial

experiments

indicated

limitations

continuous tracking when using a restricted field of view as tracking would be lost when larvae move out of the frame. The Multiscope optics was modified to reduce the system magnification and

identity tracking and therefore making it difficult to extract any features of interest. This was improved by enlarging the field of view and decreasing the number of larvae in the well, though this did not fully resolve the issue of identity tracking. It was

Figure 2. An image of Platynereis dumerilii captured by the Multiscope, showing the larger, low magnification field of view.The smaller field of view from the high magnification system is shown inset.

often the case that TRex failed to track the larvae,

Overall, by refining the optics and image processing

so the identities of the larvae in each frame had to

we were able to successfully identify, process and

be edited manually and the data had to be extracted

analyse more larvae tracking data throughout the

after this time-consuming process. Manually editing

experiment.

identities also introduced a bias towards selecting

Reflections

and tracking larvae which remained relatively still. Putting fewer larvae in each well (e.g. nine larvae compared to the previous 18) proved to make tracking and the subsequent analysis much easier. Additionally, it was identified that the number of frames per second (fps) was low for accurate tracking. The rate of 1 fps meant that each well was imaged with a nine-second interval. As a result, there was significant movement of larvae between some frames, which hindered the tracking abilities of the software.

ISSUE 65 MARCH 2022

Without the support from the Royal Microscopical Society and Dr Alex Corbett, I would not have had the opportunity to learn more about microscopy, image processing techniques and computational analysis used in research. I enjoyed working in fine detail while investigating complex biological systems and gained a deeper insight into how optical instruments function, both theoretically and experimentally. Having had little experience with practical physics and laboratory work due to the pandemic, the project gave me an opportunity

Figure 3. Using ‘TRex’ to analyse the obtained data.

to appreciate the experimental aspect of physics

One of the main highlights of my time in Exeter

more while encouraging a deeper interest in

was becoming a part of the research community.

optics. Additionally, the project developed my

I was given an opportunity to work with leading

programming skills while working with new Python

researchers at an institution I had never previously

modules for image processing and tracking. Using

visited. Attending talks, visiting laboratories and

the openly available software such as ‘TRex’ allowed

reading relevant research papers gave an insight

me to work with some of the more complex tools

into how physics is developed and applied beyond

available for scientific research.

lecture theatres, teaching me some of the skills necessary to become a researcher.

Figure 4. Speed distribution of manually selected larvae pre and post feeding.

References [1] Ashraf M, Mohanan S, Sim BR,Tam A, Rahemipour K, Brousseau D, Thibault S, Corbett AD, Bub G, 2021. Random access parallel microscopy. eLife 10, e56426. https://doi.org/10.7554/eLife.56426. [2] Williams EA, Jekely G. 2016. Towards a systemslevel understanding of development in the marine annelid Platynereis dumerilii. Current Opinion in Genetics & Development 39: 715-181. doi: 10.1016/j. gde.2016.07.005. [3] Conzelmann M, Williams EA, Krug K, FranzWachtel M, Macek B, Jekely G. 2013. The neuropeptide complement of the marine annelid Platynereis dumerilii. BMC Genomics 14: 906. https:// doi.org/10.1186/1471- 2164-14-906. [4] Walter T, Couzin I. 2021.TRex, a fast multi-animal tracking system with markerless identification, and 2D estimation of posture and visual fields [5] Baker, M. 2016. Reproducibility crisis. Nature, 533(26), pp.353-366.

Future plans Cross-fertilisation between biology, physics and mathematics allows researchers to interrogate biological complexity at all scales, from studying cells to populations and organisms, and during the next few years I hope to further pursue studies in biological and soft matter physics through theoretical, computational and experimental tools. Imaging of biological systems and structures plays a crucial role in driving biophysics research and I am grateful to the Royal Microscopical Society for funding this opportunity and Dr Corbett for his support and guidance. I have gained a deeper understanding of the topic that will most likely be a major part of my studies in the upcoming years. Elene Lominadze

ISSUE 65 MARCH 2022

Crystal of low-sodium salt (sodium-potassium chloride), magnified 25x Revital Katznelson Chosen as the December image for the 2021 RMS Digital Calendar.

NEWS

From the RMS President

The arrival of the new year may now be well behind

RMS, and this year was no exception, with several

us, but since this is our first issue of infocus for

important online meetings and events covering

2022, I would like to take this opportunity to wish

multiple branches of microscopy. These included

you all a belated Happy New Year and a peaceful and

Light Microscopy Facilities Meeting (LMFM) and

prosperous 2022 filled with good health!

Flow Cytometry Facilities Meeting, each attracting

The month of January is usually a busy one at the

approximately 300 attendees over two days,

ISSUE 65 MARCH 2022

and EM-UK 2022 – a key fixture in the Electron

from Surface Plasmons to Space-Time Resolution

Microscopy calendar – with over 130 participants.

Using EELS’. This Lecture Series is such an exciting

EM-UK and LMFM had initially been scheduled to take place in London and York, respectively, marking the long-awaited return to in-person RMS events. We were all hugely disappointed when the arrival of the Omicron variant in December meant we had to quickly re-adjust, which was possible due to our highly experienced RMS Staff and members! As we have all experienced on many occasions now, Covid-19 has no regard for best-laid plans. However, our virtual offerings in January once again demonstrated the value of the online format in facilitating a truly international audience, providing access to many people who would not otherwise be able to attend in person. Speaking of Covid and the emergence of the Omicron variant, there are encouraging signs the pandemic may finally be ‘loosening its stranglehold’, heralding the possibility of a return to a ‘new normal’ for many people. Of course, the global picture is complex and precarious - not least the result of unequal access to vaccines, which is still a major issue, in tackling Covid-19 itself. It may yet be some time before we genuinely ‘learn to live’ with the virus as an endemic reality, but at the very least, there is reason to be optimistic about 2022. I sincerely hope that we will be able to meet again in person soon. I hope many of you have been able to enjoy the presentations in the International Microscopy Lecture Series, which kicked off in October 2021 and continues to go from strength to strength. Alongside our partner organisations, the Microscopical Society of Canada, the Israel Society for Microscopy, the Brazilian Society for Microscopy and Microanalysis, and IFSM, we were delighted to welcome presentations from Professor Ray Egerton in December 2021, and Professor Ricardo Henriques in January, 2022. As I write, we are eagerly anticipating our 15 February talk from Professor Philip Batson, entitled ‘The Road

collaboration, bringing together societies around the world with the shared aim of advancing science and microscopy on the international stage as well as providing a unique educational platform, and highlighting pioneers in our field. We hope to expand this concept further and work with other microscopy and microanalysis societies and organisations to establish a truly global initiative. If you have not already done so, please visit the RMS website (www.rms.org.uk) to find out more about this collaboration, including recordings of some of our past speakers in informal conversation about their careers and their perspectives on microscopy. I am delighted that the Society’s Outreach and Education activities are now fully back up and running – including the Microscope Activity Kit (MAK) scheme for primary schools across the UK and Ireland. It is great to receive the feedback from schools benefitting from this very important initiative; and it is vital that we can continue to provide this resource that uniquely introduces students to the wonders of science made possible by microscopy. Hopefully, we will see the resumption of public outreach events later this year, bringing microscopy to yet more children and families, and inspiring people to take a keener interest in the sciences. In this issue, you can also read some of the reports provided by our 2021 Summer Studentship recipients, most of whom were able to conduct their projects in person. It is particularly pleasing to see what these students have been able to achieve, and a reminder – if one was needed - that the future of microscopy is in safe hands. Applications for our 2022 Summer Studentships are now open, with all the information available at www.rms.org.uk Here’s to a great 2022 – both in life and microscopy! My warmest wishes to you all. Professor Grace Burke, RMS President

NEWS

Welcome Jade Sturdy! A very warm welcome to new starter Jade Sturdy,

Jade said: “Everyone at the RMS has been so friendly

who has taken on the role of PA to the Chief

and welcoming and I am looking forward to getting

Executive.

to know everyone better over the coming months.”

Prior to joining the RMS Jade worked as an Executive Assistant and Governance Support Officer for the Berks, Bucks and Oxon Wildlife Trust. In that role she worked closely alongside the different committees of the charity – experience which holds her in good stead for her new position at the RMS. Jade has three children who keep her very busy outside of work, but when she does have some time to herself, she enjoys baking, adapting recipes and coming up with new creations.

Summer Studentship applications open Applications for the RMS’s fantastic Summer Studentship scheme are now being invited. Up to six studentships of £2,000 are offered every year, split evenly between physical sciences, biological sciences and interdisciplinary projects. Applications for our Summer Studentships must include a significant microscopy component and should be submitted by a suitable host academic on behalf of a student

Rana Salem was among the successful applicants for an RMS Summer Studentship in 2020.

The deadline for applications is 31 March 2022. The Studentship is offered on the understanding that a 500-word project report is completed by the student by the end of the period of study and submitted to the RMS (you can read some

second year of study for a three-year degree, or at the end of their second or third years for four-year degree courses. Master’s students are not eligible.

of last year’s Summer Studentship reports in this

The person making the application must be a

issue). Students will also be asked to do a two-to-

Member or Fellow of the RMS.

five minute recording briefly talking about their

Find out more and apply at www.rms.org.uk

experience for the RMS YouTube Channel.

Awards will be made to students at the end of their

ISSUE 65 MARCH 2022

A fond farewell to Events Organiser Kat Driscoll The RMS team recently said a warm goodbye to

and her positive attitude has rubbed off on us

Events Organiser Kat Driscoll, who is leaving to

all. We will all miss her greatly, but it’s fantastic

pursue her ambition to become a primary school

that she is pursuing her teaching ambitions, and

teacher.

we wish her the very best of luck in her future

Kat has worked on some of the biggest RMS

career.”

events since joining the Society just over two years ago. She was instrumental in delivering the successful Microscopy and Microanalysis in Geological and Archaeological Sciences meeting last autumn, and UK Light Microscopy Facilities Meeting (LMFM) 2022 in January. Kat will begin a PGCE course at Exeter University this September, and will be working as a Teaching Assistant to gain further experience over the next few months. RMS Chief Executive Allison Winton said: “Kat has been a really popular member of the team

Apply now for an RMS Award! Applications are now open for a wide range of

the 'unsung heroes' of microscopy by making an

prestigious RMS awards covering all aspects of

award to an engineer, technician or laboratory

microscopy.

research support scientist)

The awards cover Scientific Achievement, Research

•

Support, Outreach and Education and more. Visit www.rms.org.uk to find out more about

2023 RMS Section Awards (awarded by each of the RMS Scientific Sections)

•

2022 Chris Hawes Award for Outreach and

each of the awards listed below, including eligibility

Education

criteria and how to apply.

who have made a substantial contribution to

•

2022 RMS Scientific Achievement Awards (awarded in recognition of the outstanding

2022 RMS President’s Award (recognises an

scientific achievements of established, mid-

exceptional voluntary contribution to the

career researchers)

work of the RMS) •

individuals

over the course of their career)

2022.

•

(recognises

education, outreach and/or public engagement

The applications deadline for each award is 22 April

The list of awards is as follows:

award

•

RMS Honorary Fellowships

2022 RMS Vice-Presidents’ Award (recognises

NEWS

Watch the International Microscopy Lecture Series on YouTube If you missed any of the opening talks in the

its initial broadcast, which means there’s no need

International Microscopy Lecture Series, you can

for anyone to miss out!

now watch them in full on the RMS website – via the Society’s YouTube channel. Each virtual lecture is uploaded around a week after

Recordings of each of our speakers in informal conversation are also available. These are uploaded in advance of each lecture. The International Microscopy Lecture Series was jointly established by the RMS, the Microscopical Society of Canada and the Israel Society for Microscopy. The Brazilian Society of Microscopy and Microanalysis has also recently joined the collaboration.

Supported by the International

Federation of Societies for Microscopy (IFSM), the lecture series is part of an international networking platform for expanding knowledge, sharing new developments, and exchanging best practices in microscopy. Find out more at www.rms.org.uk

RMS Council Nominations Nominations for future Council Members will be

Trustees (also on the Charity Committee website)

proposed and agreed by Council at their Spring

and section committee members.

meeting, and published in the RMS AGM Agenda on

RMS Section Committee Nominations

the RMS website soon after. Members can also nominate members of Council. At

Committees must be received by the Chief

any date not less than three calendar months before

Executive of the Society, in writing, at least 28

the Annual General Meeting, any four members may

days before the Section Annual General Meeting,

nominate any other duly qualified person to fill any

and must be supported by at least two members

of the offices of President,Vice-President, Honorary

of the Society. The consent of the nominee must

Secretaries, Honorary Treasurer or other member

be obtained. The RMS is committed to being

of Council by delivering the nomination in writing

a welcoming, inclusive Society and encourages

to the Chief Executive, together with the written

diversity across all activities and in the membership

consent of the nominee to accept office if elected.

of our committees and groups. All nominations

A copy of the Society’s Royal Charter and By-Laws is available online, as well as a full list of current

Nominations for election to any of the Section

ISSUE 65 MARCH 2022

should be sent to RMS Chief Executive Allison Winton awinton@rms.org.uk

Journal of Microscopy: Early Career Special Issue – call for papers! Submission deadline: 6 May 2022 A Journal of Microscopy Special Issue is in the pipeline featuring recent microscopy developments by Early Career Researchers.The issue will be titled A Lens on the Future: Next Generation Microscopy by Next Generation Microscopists and submissions are welcome from any microscopy discipline. The issue is being guest edited by Dr Liam M.

Rooney (Heriot-Watt University, Edinburgh) and Dr Laura Clark (University of Leeds)

Eligibility First authors (or last authors for Early Career PIs) must be identified as Early Career Researchers.This is classified as any person undertaking work in the field of microscopy/flow cytometry and belonging to one of the following categories: • Current undergraduate / postgraduate / Masters / PhD students; or • Within eight years of starting work or studies in a microscopy-related field (excluding career gaps) (i.e. eight years after leaving education) • *Submissions from outside of the above criteria will be considered on a case-bycase basis by the Guest Editors. Author guidelines can be found on the Journal website and the submission deadline is 6 May 2022. If you have any questions, please contact the Journal Editorial Office Manager, Jill Hobbs journaladmin@ rms.org.uk

NEWS

‘God Bless the Microscope!’ – RMS History available to download on RMS website Read Gerard L'E Turner's superb historical work, first published in 1989 The definitive account of the first 150 years of RMS history is now available to download. Written by the late Gerard L'E Turner, RMS President from 1974-75, God Bless the Microscope! was first published in 1989. Beautifully written and illustrated, the book charts the development of the RMS from its origins as "The Microscopical Society of London" in 1839, through to the late 20th Century. The text covers all the key milestones in RMS History and brings to life some of the most important figures - and colourful characters - responsible for shaping the work of the Society over the decades. Visit the history pages at www.rms.org.uk to download a copy. Also available to download is Dr John L Hutchison’s ‘Moving Forward’ - a brief outline of the developments within the RMS from 1989 - 2014 produced for the Society’s 175th Anniversary celebrations.

January RMS meetings provide great start to 2022 A huge ‘thank you’ to everyone who took part in our first virtual meetings of 2022 and helped make them such a success.

Our January events covered a wide range of microscopical methods, techniques and applications, drawing in attendees from across the world. The UK Light Microscopy Facilities Meeting (LMFM) and Flow Cytometry Facilities Meeting each attracted around 300 participants over two

ISSUE 65 MARCH 2022

days, and more than 130 people logged on for EM-UK 2022. The latter event had initially been planned to take place at London’s Natural History Museum - but was swiftly moved online amid concerns over the Omicron Covid-19 variant. We very much hope to see the return of some inperson events later this year. As always, please check out the online events calendar at www.rms.org for all the latest information on upcoming RMS meetings, courses and conferences.

Ian Titley steps down from infocus upon his retirement At its December meeting,

the

infocus Editorial Board

bade

fond farewell to Flow Cytometry re p re s e n t a t i ve Ian Titley, who is due to retire from his role as

• What have you enjoyed most about being part of the infocus team? Working with the editorial team and the RMS colleagues who support the editorial board. It is always a stimulating experience and great to have enthusiastic members who are always striving to improve the content of the magazine. • What is your fondest memory of being part of infocus?

Manager of the Sutton Flow Cytometry Facility at

I thoroughly enjoyed the 50th issue article by Aude

the Institute of Cancer Research later this year.

Mongiatti concerning microscopic analyses of

Ian has been a key member of the editorial team

Scythian gold by members of the British Museum.

for the last eight years, sourcing articles and other

• What have the biggest challenges been

content for our readers, and helping shape the future of the magazine.

for the editorial team during your time on the board?

Everyone at infocus and the RMS would like to

Always the ‘article pipeline’, that is, ensuring we

thank Ian for all his contributions, and wish him a

have enough copy to fill the next issue. I have to say

long and happy retirement.

during my time on the board the well has never run

As a parting gesture, Ian kindly answered a few

dry, which is a great testament to all the members

questions about his time at the magazine, and its

of the RMS who willingly, and with no financial

place within the microscopy community:

reward, submit articles.

• What do you think are the best

• When are you due to retire and what

aspects of the magazine, and why?

do you plan to do in your retirement?

My view of Infocus is that it should be a light but

I retire from my full-time post at the end of March

informative read and in part facilitate the social

2022. I will then fill in two days per week until the

aspects of the RMS. For me, meeting reports

end of July 2022 when I will fully retire. I have some

(remember those?) are very good, news of new and

photographic kit that is very underused, so I plan

old members, and since light microscopy is visually

to rectify that.

rich, it is always a feast for the eyes.

• Will you still try to attend microscopy

• Why is a magazine like infocus so important for the microscopy community?

/ cytometry events and keep up with the latest developments in the field? We shall see, I think once one is out of the

It circulates news of the actions and events of

professional scene it is difficult to participate but I

RMS and provides entertaining and thoughtful

might pop up from time to time, particularly in the

articles about many aspects of the varied forms of

London area.

microscopy.

Microscopy in the Third Age – You are never too old to Learn! Michael Gibson

ISSUE 65 MARCH 2022

For as long as I can remember I’ve been involved with microscopy in one way or another. As a teacher I enjoyed the opportunity of being able to encourage youngsters of all ages to effectively use microscopes in the classroom setting. When I got older my interest was sustained and enhanced by membership of various clubs and societies including the RMS. Now that I am retired, I still find that much of my time is spent looking down microscopes, preparing slides, writing articles, attending meetings and doing outreach work particularly in schools, libraries and youth clubs.

Figure 1. Epitheilial cheek cells x40.

During the last few years, I have also been part

a good deal of autonomy in setting its own agenda

of an organisation known as the University of the

and learning styles in accordance with what best

Third Age (u3a). Founded in 1982, u3a is a UK-wide

suits both the subject and the participants.

movement of regional “educational co-operatives” that provide a wide range of opportunities for retired people to meet, learn and have fun. Members explore new ideas, skills and activities together in various small-group activities that range from art, astronomy, photography, history, philosophy and science, through to such subjects as music, jazz, card games, table tennis, walking and cycling - in fact anything you can think of.

When I first joined Northampton u3a there were over one hundred different activities to choose from, but sadly no microscope group. After some discussion with the organising committee, I was given the go-ahead to set up a microscope group which in fact turned out to be the first one in the country. It quickly attracted a number of members, keen to learn new skills and some to add to their existing knowledge and experience of microscopy

One of the key ideas u3a is based on is the principle

gained whilst in full-time employment. However, one

of “self help” and might best be described as those

initial problem encountered was not just recruiting

who learn shall also teach. Each interest group has

new members, but also finding a suitable place to

ISSUE 65 MARCH 2022

Figure 2. Onion cells.

Figure 3.Tracheid element.

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meet. Fortunately, as I was already a member of our local Northamptonshire Natural History Society, we were able to use their rooms, facilities and microscopes for our monthly meetings. From the outset we emphasised that this is a beginners’ group and therefore no previous experience or knowledge was necessary; the essential idea was to discover, learn and have fun. Currently there are between twelve and sixteen regular participants, many of whom now own their own microscopes - some new and others bought second hand on eBay and elsewhere. Our varied programme includes a mixture of practical activities, discussion, and slightly more formal lecture-style presentations, including where appropriate, online videos covering a range of microscope-related topics. Even during the last 18 months of Covid and lockdown the group managed to keep going with meetings online. In the last year alone, we’ve managed to incorporate into the programme such topics as “indoors and outdoors with your microscope”, “making slides and mounting methods”, and “forensics and the microscope”. In April through to July this year some of the members have participated in a citizen science “Nenescape project”, sampling and recording microscopic life found along various stretches of the River Nene, around Northampton. When using our microscopes, recording what we do and see is important and therefore it’s not surprising that photography features in many aspects of practical work. Although some of us use fairly sophisticated photographic equipment, quite good results have been obtained by members just using their mobile phones in afocal mode either fixed or hand-held and positioned over the microscope eyepiece. I’ve included here a selection of some of the photographs that have been taken in our studies and practical work over the last couple of years. Fig 1 shows a photograph from our earliest work looking at squamous epithelial cheek cells and

Figure 4.TS Pine stem with resin duct NEG3x40obj.

ISSUE 65 MARCH 2022

Figure 5. Bindweed leaf peel.

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Figure 6. Collagen fibres in leather sample.

Figure 7. Diatom strew 6 phase x40.

Figure 8. Cladophora x40.

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making simple slides with methylene blue. In this example using a x40 objective lens, it was possible to make out the plaque bacteria that often form as a biofilm on the cell membrane. This led onto further work by some of our members in making their own bacterial slides from yoghurt. In Fig 2 we managed to get a very good Low-mag image of onion cells, here stained with ordinary food colouring. Fig 3 is an image from a macerated sample of toilet tissue showing a single xylem element found amongst the wood fibres stained again with food colouring. Fig 4 shows part of a pine stem in transverse section illustrating one of the resin ducts from a prepared Biosil slide. In Photoshop we experimented with the original brightfield image to produce this effect in negative that is reproduced here. In one session we had great fun making leaf peels which is a relatively simple technique using clear nail varnish painted over part of the leaf surface. Once dry, the varnish is peeled off using Sellotape and mounted onto a slide. Fig 5 shows the final result with the underside of a Bindweed leaf seen here with Rheinberg illumination. Two years ago, before the pandemic and lockdown, we were approached by the museum of leathercraft

Michael R. Gibson Michael Gibson is a health educator and

in Northampton, requesting us to photograph and

retired teacher with a lifelong interest in and

catalogue many of their microscope slides. Here in

involvement with microscopy. He belongs to

Fig 6 is a photograph using cross polar illumination

several related societies including the Quekett,

showing the collagen fibres in a prepared sample of

RMS, the Postal Microscopical Society and the

leather from their collection.

Northamptonshire Natural History Society. He

Finally, I’ve included further images from our

has written a number of articles on various

collaboration this year with the lottery-funded Nenescape Project (“What’s in that Monster Soup?”) involving some of our members in a survey of diatoms and other phytoplankton found along various stretches of the River Nene around Northampton (Figs. 7 and 8). This project was undertaken with help from members of the Quekett Microscopical Club.

subjects including photography, microscopy and health education. As an educator, Michael has a passion for making the invisible visible through the use of camera and microscope, and of helping others develop both their scientific knowledge and understanding of the scientific world in all its wonder and beauty as revealed by the microscope.

Lynne Joyce interview In this issue, infocus speaks to biologist Dr Lynne Joyce about her career in science and microscopy – including her time at the Lord Rank Research Centre and Agar Scientific Ltd. Lynne has a long association with the RMS, and served as Honorary Treasurer for 16 years. Now retired, she is still closely involved with the Society, and sits both on the RMS Council and History Committee. Let’s find out more! Lynne was born in Tynemouth in the North East

only male teacher was the physics master. There

of England, her early childhood coinciding with the

were only five of us in the class. The lessons seemed

post-war years. Her father, who had served in the

full of equations and perhaps not terribly appealing.

RAF, was the director of a company which sold

I now know better and should have tried harder!”

Morris motor cars, while her mother worked at the local hospital.

Having completed her A-levels, Lynne went on to study biology at York University – though at the

“I was an only child and probably a little bit spoiled”,

time of applying, she had yet to form a clear idea of

says Lynne, “but I was very lucky because my parents

a potential career path, or even her next immediate

believed in getting a good education. I remember my

steps.

mother used to do all the cooking, which is probably why I never learned to cook very well.”

She recalls: “When I left my school, going to university was expected of you. Most of us either

It was on her daily walk to primary school that

went into teacher training or university, and I really

Lynne began to take a keen interest in the natural

didn’t know what I wanted to do. In the end I applied

world around her. She explains: “I used to walk past

for the Biology Department at York.The department

a garden with nasturtiums which always seemed to

had a very good microscopy department, with Tony

be covered in caterpillars. It was the way they moved,

Robards there at the time.”

and how they had decided to live on these lovely orange nasturtiums - probably because they had

Lynne soon became attuned to the daily academic

munched all the leaves! I suppose that’s one of my first memories of really taking an interest in biology. “For secondary school my parents sent me to a convent school which had a reputation for achieving good results – a train ride from home - though I’m not a Catholic. I didn’t really gel with languages, history and geography, but I did enjoy the botany, zoology and chemistry. The domestic studies were not my scene, so it may have simply been a process of elimination, rather than a choosing process.” She adds: “I really didn’t like physics very much – perhaps because it was an all-girls school and the

ISSUE 65 MARCH 2022

Dr Lynne Joyce

Lynne taking a call at the Agar office in the 1980s. No computer required!

demands of studying for a science degree, and

firstly in the Crop Science Department, and later

also gained her first serious experience of using

in the Electron Microscopy Unit. It was an exciting

microscopes. Like many science students before

opportunity – though not without an element of

and since, she occasionally looked on enviously at

trepidation.

the less structured timetables of her arts-studying counterparts!

Lynne recalls: “Moving somewhere as far away as High Wycombe was quite a wrench in those days,

She says: “As a biology student, unlike the arts, you

but it was very difficult to get jobs in the North East,

went to lectures first thing in the morning and then

with so much unemployment at the time. If I had

you did the practical sessions, so you were occupied

gone into the teaching profession, that would have

the whole day. I became more interested in plants

been a bit easier. It took me more than 18 months

than animals because I couldn’t bring myself to kill

to write up my PhD – not just because I had started

the mice for the dissection practicals, and I had to

working by then, but because there were children

bribe the demonstrators to do it for me! I quickly

living in the house where I was renting a room, and

realised that the plants were going to be a lot easier

they used to come into my room when I was at work

to cope with.”

and regularly re-arrange my papers!”

By the early 1970s, Lynne was studying for her

She adds: “It was a relief that I had got a job, and a

PhD on ‘The Behaviour of Plant Cells in Culture’ at

bit scary that I was out in the big, wide world, but I

Newcastle University’s Department of Plant Sciences

was excited by the research and working with such

in the Faculty of Agriculture. She was still writing her

a wide range of people. I was in the Crop science

thesis when a job opportunity came up at the Lord

department with regular contact with the engineering

Rank Research Centre at High Wycombe (which,

and bakery departments and the microscopy unit.

as infocus readers may recall from our June 2021

My initial research was trying to produce haploid

issue, was also where the respected food scientist,

plants from pollen grains in tissue culture.There was

Roger Angold honed his skills). Lynne would go on to

also the production of Quorn – the meat substitute

work here for 10 years as a Senior Scientific Officer,

- going on at the time.”

So what did Lynne enjoy most about working at

not at the higher level. I never felt uncomfortable or

Rank Hovis – and the microscopy in which she was

experienced any discrimination.

engaged?

“In a way I think I have been very lucky throughout

“I think it was just seeing how everything worked

my career in that I have always been considered first

together from a microscopy level to a ‘whole’ level”,

and foremost as a person, and there were never any

she explains. “I loved looking at the TEM of the

issues. I just thoroughly enjoyed all my time there,

wheat grain and the structure of bread and doughs –

and the contribution that microscopy made to the

thinking about all the structures and how everything

food industry.”

works together. “I worked alongside Roger Angold and Mick Williams

Agar Scientific

who were very innovative.We had a TEM and SEM and

After leaving RHM Research, Lynne’s next big career

probably one of the first cryo-systems. I remember

break came when she moved to Essex with her

sitting there watching the liquid nitrogen puffing out.

husband Doug (a plant scientist who was also at

It was really interesting to see the structure of foods

the Research Centre, and whom she met during her

but also, getting involved if there was a problem on

post-graduate years at Newcastle).

the production lines. I remember going down to Kent to do some work on the production line, where they were brewing up great vats of starch - so different to the laboratory environment.” While women’s representation across the sciences – both in academia and industry – has increased over the decades, the landscape in the 1970s was of course very different, as, it’s probably fair to say, were the prevailing attitudes of the time. But what was life really like as a female scientist in the 1970s? Did Lynne ever feel uncomfortable, or even experience any discrimination first-hand?

She says: “He got a different position, but still with one of the Rank Hovis companies. I worked for him doing field trials, and also in a commercial seed lab doing germination trials. “Fairly soon after that, I met the wife of Fred Sheldon, she made filaments at Agar Aids (now Agar Scientific), and they were looking for somebody to cover work in the lab because their technician had gone to New Zealand for a year. I had experience of using Agar filaments and consumables in the microscopy unit at RHM, so I went for an interview and got the job. Alan Agar was a physicist and Fred was a material

She says: “In the Crop science department, there

scientist, so I was the biologist that joined the team.

were two women in senior positions – a plant

It was a case of being in the right place at the right

pathologist and myself – and there were quite a lot

time.”

of technical female staff. Certainly at the technical level there was probably a good mix, but perhaps

Lynne stayed on at Agar after her temporary position came to an end, moving away from hands-on microscopy and taking up a role as Customer Liaison Officer and Sales Director. Her responsibilities included

improvement

customer

service,

technical writing, queries and quotations. She also took on responsibility for the mail order catalogue. In 1989, after Alan Agar retired, she was appointed Sales Director with responsibility for promoting sales into the U.K. and export markets. Three years later she became the company’s Managing Director Lynne in action at the Agar exhibition stand.

ISSUE 65 MARCH 2022

– a position she held until 2008 when she took on

Lynne with Alan Agar at MICRO '94.

the role of Director of Market Research until her

introduced me to many people. He said he couldn’t

retirement seven years ago.

introduce me because he couldn’t always remember

She says: “My time at Agar gave me such an amazing

names – he knew so many people!”

experience and insight of the whole microscopy

Lynne went on to serve for 10 years as RMS

community and industry. Alan originally started

Honourary Treasurer from 1995 to 2005, and was

the company as a consultancy, but the economic

also a member of the Society’s Trade Advisory

climate at that time was not good so he decided

Committee (now the Corporate Advisory Board -

to manufacture the filaments and the calibration

CAB) from 1992. She served a further six years as

specimens that microscopists required. He put

Honorary Treasurer from 2014 to 2020, and is still

together a range of items that were needed in order

an ‘invited’ RMS Council member. She also sits on the

to make a specimen to look at in the microscopes

Society’s History Committee.

– fixatives, resins, tweezers, glass used for making ultra-microtomes, knives – and also importantly, free technical advice. At that time there was a lot of people working by themselves, and they just needed advice on how to do things.”

Lynne at the RMS It was also Lynne’s move to Agar that led to her involvement with the RMS. She recalls the first conference and exhibition she attended – a European meeting held in York in 1988 - on the Agar exhibition stand. “I saw so many of the people I had spoken to on the phone and also met many microscopists and students from all disciplines from UK and Europe – it was such an experience. I also got to meet a lot of the other exhibitors. “Afterwards I told Alan that I thought I mustn’t have been doing my job very well, because he hadn’t

Reflecting on her long association with the Society, Lynne says: “I was very lucky because both Alan and Fred gave me time off from Agar to spend time on RMS business, because they were both very supportive of the RMS and that was important. I have been able to see the RMS grow and progress, which has been exciting and rewarding. One of the key events during my first period as Treasurer was the end of the MICRO meetings taking place in hotel basements, and the change to the MicroScience exhibition and Conference at London’s ExCel in 2002.” She adds: “With Agar, I was also going to exhibitions abroad, so I was able to see what microscopy conferences in other countries were doing and that helped to inform both the CAB and the RMS as a whole – because it is obviously an international society. I have found it so interesting working under the various RMS presidents. It’s been a real eye-

opener to see how they have moved things forward

windscreen would be completely covered in flies.We

and made the society what it is today.”

also used to have frogs and newts in the garden. On

So what, for Lynne, are the most important aspects of the Society’s work, continuing to this day? “I think the age range and diversity of its members are really important, as are the Science Sections, Focused interest Groups, Bioimaging UK, conferences and exhibitions. There’s also the one-day meetings and training courses - plus the Outreach activities and microscope kits for schools. The Society is always keen to embrace new challenges and the staff have been brilliant in in meeting those challenges. She adds: “The virtual meetings over the last two years have done an enormous amount for the RMS because it has enabled us to relate with more microscopists, both domestic and international – people who wouldn’t normally be able to travel to an RMS event. We have obviously missed ‘in-person’ meetings but I have to say I have also been able to attend more meetings than I probably would have done.”

in the local area, so things do also just change over time. The garden birds have also changed over the years.” Reflecting on advances in microscopy which have occurred since her time as a ‘hands-on’, working microscopist, Lynne looks on in wonder – and perhaps even with a tinge of envy – at the technological improvements and evolving techniques at the disposal of today’s microscopists engaged in research. She says: “Of course, when I was working, the resolution wasn’t quite as good as it is now. When a big technological advance is made, there are some people in microscopy who will say ‘I predicted that would happen 20 years ago’, but I was just totally amazed at how much better it got, and how much microscopy has contributed to everything in everyday life. And these days, to sit in meetings with some of the Early Career people who are so passionate

Walks, wildlife and words of advice

and excited by microscopy, it is just wonderful to

Still based in Essex, Lynne and her husband enjoy

She adds: “Some things tend to plateau, but with

walking, gardening and keeping an eye out for the local

microscopy, it just seems to go up and up – both in

wildlife. She is a member of her local horticultural

terms of the instrumentation and automation. The

society, as well as the Alpine Garden Society, while

time we used to spend aligning the instrument and

her husband is a keen photographer and a member

the time to just prepare a specimen, and now you

of the Suffolk and Essex Wildlife Trusts.

just press a button and it is just magical. In some ways

She says: “When we first moved to the countryside

I’m a bit jealous that I didn’t get to do hands-on stuff

know that this is going to continue.”

I was still working fairly long hours, but I always had

with all this modern technology!”

a strong feeling that if you live in a village, you should

And what words of advice does Lynne have for

be giving something back to the village. That’s why I

anyone starting out in science and microscopy?

became involved with the horticultural society.”

the other hand, we now have red kites and buzzards

“I would say ‘just do it’ and take opportunities when

Over the years, one thing she has witnessed first-

they come up”, she says. “Things are a lot more

hand – and become concerned about – is the

structured these days, and it has also become much

reduction in wildlife around the local area.

more international, so you can get experience in

She says:“If you just look around the countryside, the

many different laboratories. So I would say just take

actual lack of wildlife is quite apparent. I remember

every opportunity that you can.”

when you used to drive in the summer, and your

Owen Morton

ISSUE 65 MARCH 2022

At the interdisciplinary heart of the matter: The RMS Engineering and Physical Sciences Committee Roland Kröger, EPS Committee Chair

The versatility of advanced microscopy and spectroscopy is demonstrated by the wide range of applications that span across the Materials Science/Life Science boundary. It underpins the push for high-resolution correlative, three-dimensional and time resolved characterisation of organic and inorganic as well as composite materials that play key roles in the development e.g. of new drugs and medical treatments as well as environmentally friendly and energetically efficient alternatives to current material systems and the cultural heritage. This addresses some of the most urgent topics of our society such as sustainable health care and climate change and provides important contributions to the protection of cultural artifacts.

The Engineering and Physical Sciences Section

in the engagement of our members in RMS events

embodies the broad and interdisciplinary engineering

such as conference sessions, workshops, seminars

and physical science community, that uses advanced

and training activities. We regard state-of-the-

microscopy - and complementary spectroscopy-

art microscopy and spectroscopy as key tools to

based techniques to address hot topics including

address exciting interdisciplinary questions, which

Energy and Energy storage, Biomaterials, Structural/

is reflected in numerous events including various

Geological materials, Advanced 3D manufacturing,

sessions at the MMC conference series, workshops

Photonic Materials, Electronic Devices, and Surface

on focused ion beam and X-ray based tools, a recent

Engineering. We have a diverse range of expertise

event on Microscopy and Microanalysis in Geological

represented by our committee members including

and

Materials Science and Engineering, Bioengineering,

org.uk/rms-event-calendar/2021-events/microscopy-

Physics, Chemistry, Geology and Archaeology.

microanalysis-geological-archaeological.html) and our

Our committee has a strong linkage to industrial

involvement in the RMS International Microscopy

partners by including company representatives from

Lecture

Health Care, Chemical Technologies and Electron

collaborate/the-international-microscopy-lecture-series.

Microscopy (e.g. previously Smith and Nephew,

html). The overwhelming participation of UK-based

Johnson Matthey and Hitachi and currently Carl

and international researchers in our events make

Zeiss and Sandberg LLP). Our diversity is reflected

research activities in our field visible beyond the

ISSUE 65 MARCH 2022

Archaeological

Series

Sciences

(https://www.rms.

(https://www.rms.org.uk/network-

membership of the RMS promoting microscopy and

interest

spectroscopy-based science. Our members engage

characterization in 3D he is particularly focussing

multi-lengthscale

material

in outreach activities e.g. by offering microscopy

on in situ techniques to study mineralisation

kits to schools and introducing pupils to stimulating

processes in liquid environments.

ways of exploring the microscopical world (https:// www.rms.org.uk/network-collaborate/science-sections/ outreach.html). Via this direct interaction with the youngest members of our society we aim to shape

Dr Anna Baldycheva (Section Deputy Chair) Anna is an Assistant Professor in

engineering and physical science of the future and

2D Optoelectronic materials in

encourage particularly girls and members of ethnic

Engineering at the University of

minorities to choose this exhilarating research field

Exeter. Dr. Baldycheva’s research

to achieve increased representation.

group works in the areas of 2D

With this scope our committee is well-placed within the RMS community and reaches out to other committees for co-organisation of interdisciplinary events within the framework of the RMS and beyond.

Materials, Si

and

Microfluidics. The research interests span from the development of new 2D material based layered and liquid crystal nanocomposites to the engineering of integrated

EPS Committee Members

Photonics

material-Si

hybrid

electronic-

photonic devices for application in communications, energy harvesting, and bio-chemical sensing. Since

Professor Roland Kröger

2010, Dr. Baldycheva authored and co-authored

(Section Chair)

over 50 peer-reviewed papers and conference Roland is a Professor at the

proceedings.

Department of Physics at the University of York concentrating on Nano- and Biomaterials

Dr Trevor Almeida Trevor is a Lecturer in the

using electron microscopy as

Materials

well as various spectroscopy

University

techniques. He obtained his PhD from the University

Materials Physics and Materials Science covering diamond

thin

films,

metal/semiconductor

nanostructures, nitride based light-emitting devices, metal nanoparticles for biomedical applications and biominerals using focused ion beam as a key method for sample preparation and analysis. Besides his

Glasgow.

obtained his PhD in Material

of Hamburg/Germany and the Fraunhofer Institute Germany. Roland has since built a large expertise in

Condensed

Matter Physics Group at the

tools including Raman microscopy and X-ray

for Surface Science and Technology in Braunschweig/

and

Science at the University of Nottingham, focusing on the transmission electron microscopy (TEM) of magnetic nanoparticles. His research evolved to investigate a range of nanomagnetic processes by combining in-situ TEM methods with Lorentz microscopy techniques. This took place during time spent at Imperial College London, Centre for Electron Nanoscopy (Denmark), Ernst-Ruska Centre (Germany), the University of Glasgow and CEA-LETI in Grenoble (France). Trevor’s primary interests include the functional magnetism

within

nanostructures,

nanoelectronics, magnetotactic bacteria, meteorites, Bone Nanostructure: Organisation of human bone nanocrystals imaged by scanning transmission electron microscopy.

minerals, nanoparticles and thin films.

Mr Ian Belding

member of the Learning Zone team at mmc and an Ian is Regional Sales Manager at

occasional contributor to infocus. His research

Carl Zeiss Ltd and Business

interests include sample preparation techniques,

Development

for

particularly those involving applications in light and

Electron and X-ray Microscopy.

scanning electron microscopy. He is currently

Ian joined ZEISS in 2004 as an

undertaking a 2nd edition of A manual of Practical

Manager

Electron Microscopy product

Laboratory and Field Techniques in Palaeobiology

specialist and has since worked in a variety of roles

(2001, published by Kluwer, now Springer). Other

across the full spectrum of ZEISS microscopy

micropalaeontological research includes a study of

products. Prior to that Ian gained a degree in Physics

the

from Leeds University and worked in a range of

foraminifera of the Tethyan Ocean recorded in

industries working on analytical instrumentation.

rocks from the NW Himalayas 50.5 million years

last

shallow

marine

carbonate-platform

ago as India crashed into Asia, Neoproterozoic agglutinated

Mr Paul Bennett-Hughes Paul works for Sandberg LLP, based in Clapham, as a Senior Associate and is involved in

foraminifera

from

Europe

(Avalonia and Baltica), and contextual studies on the world’s oldest (3.5 billion years old) putative microfossils from Western Australia.

construction materials related investigations,

such

Dr Dan Haspel

compositional analysis, staining issues,

ASR/DEF

assessments,

Since the beginning of 2017 Dan

fire-damage

has been a Technical Specialist in

assessments and general condition analysis. As part

the

of these investigations, Paul frequently conducts

Microscopy

petrographic examinations of natural and man-

University of Plymouth, primarily

made

electron

in charge of the FIB-SEM and is

materials

using

microscopy techniques.

optical

and

Plymouth

Electron

Centre

the

Paul is a European

the lab’s specialist for Materials and Engineering.

Geologist, Chartered Geologist and member of The

Dan graduated with his PhD in 2018 from

Geological Society of London’s Applied Petrography

Loughborough

Group (APG).

developing a dual-layer system for the mitigation of

University

which

looked

tin whisker growth. Prior to this, Dan obtained his MEng, also from Loughborough University, in

Mr Owen Green Owen has worked in the Earth Science Department at the University of Oxford since 1989. He initially, trained and worked in

London

Geological

Colleges

Technician

a and

Curator of Geological Collections. He is currently a member of both the Engineering and Physical Sciences and Outreach Committees, and has been a co-convenor

the

Geo-materials

meeting

(September 2014), and organised Outreach events on volcanos and mountain building. He has been a

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Automotive Materials Engineering. Dan still studies tin whiskers but now with the addition of 3D analytics using FIB-SEM. Being within a central unit, he is heavily involved with a broad range of subject areas using FIB-SEM, creating and analysing 3D datasets. Dan is also highly interested in finding novel samples to use advance analytical techniques with, such as EBSD and TKD (transmission-EBSD) of teeth. More recently, Dan, along with other Plymouth EM colleagues, have been successfully using 3D printing to create bespoke sample holders for use in electron microscopes.

Professor Beverley Inkson Beverley

Professor Igor Meglinski

Professor

Igor is a Professor in Biomedical

Nanomaterials in The Dept of

Engineering & Biophotonics at

Materials

and

the School of Engineering &

Engineering at The University of

Applied Science and School of

Sheffield, where she leads the

Life & Health Sciences in Aston

Science

NanoLAB

Centre.

Beverley

University

(UK).

joined the RMS as a PhD student in Electron

Chartered Physicist (CPhys), Chartered Engineer

Microscopy at Cambridge University, and served on

(CEng), Fellow of Institute of Physics, Senior

the RMS EM section committee while a Royal

Member of IEEE and Fellow of SPIE. His work is

Society University Research Fellow. She set up the

focused on the examination of interaction of

UK NanoFIB network in 2001, and has run

photonic quantum vortexes with biological tissues,

numerous Focused Ion Beam scientific meetings

and their components, including cells, cell’s

joint with the RMS. Beverley’s research interests

organelles, collagen, etc. He is developing an

focus on the use of electron and ion microscopies

advanced

to quantify the nanomechanical and functional

tweezers for cells diagnosis and quantitative

properties of 3D nanomaterials, including developing

polarization-based hyperspectral imaging technique

novel

for tissue characterization. His particular interest is

in-situ

SPM-TEM

and

tomographic

characterisation methods.

optical-microscopy

biopsy,

optical

an integrating of new developing technologies with currently available microsocopy systems.

Ms Nyree Manoukian Early Career Representative

Dr Duncan Muir

Nyree studied archaeology for

Duncan is the Senior Electron

her undergraduate degree at

Microbeam Technician in the

University

and

School of Earth and Ocean

subsequently moved to the UK

Sciences at Cardiff University.

for

His work focuses primarily on

of Toronto,

her

technology

MSc

degree

and

analysis

scanning electron microscope

archaeological materials. She is currently a PhD

imaging and microanalysis of geological samples.

student at the University of Oxford in archaeological

Duncan studied Geology and gained a PhD at the

science. Her research interests include the analysis

University of Bristol researching subduction zone

of ceramics using scanning electron microscopy and

volcanism and magma processes. Prior to his

polarised light microscopy, in order to reverse-

postdoctoral studies he worked in the applied fields

engineer the production and use of such materials

of Mineral Exploration and Offshore Geotechnics

in the past. Her fieldwork activities focus on the

gaining a broad experience of Geosciences.

Caucasus region. Research areas include raw materials acquisition, manufacturing techniques, firing regimen, and the use/function of archaeological pottery.

Nyree

Representative

is and

the is

EPS keen

Early on

Career

organising

archaeological science workshops, which integrate various scientific fields explored within RMS.

Dr Fabio Nudelman Fabio is a Senior Lecturer at the School of Chemistry, University of Edinburgh. Fabio obtained his PhD at the Weizmann institute of Science, Israel, and then

moved to the Eindhoven University of Technology,

modalities to further a deeper level of understanding.

The Netherlands as a postdoc before joining the

She is a Chartered Engineer and a Fellow of the

University of Edinburgh as a Chancellor’s Fellow.

Institute of Materials, Minerals and Mining. Having

Fabio’s current research is in biomineralisation,

previously obtained a Masters in Gas Turbine

investigating the formation and the structure of

Materials from Swansea University she is currently

mineralized biological materials such as bone, teeth

studying part-time for a PhD in single crystal

and shells. This is an exciting area at the interface

materials at Cambridge University.

between materials sciences, chemistry, crystal growth and biology. Cryo-electron microscopy techniques, including cryoTEM, cryoSEM and

Ms Xiangli Zhong Xiangli is a Senior Experimental

cryoFIB-SEM play a central role in Fabio’s research,

Officer

and in the last years he has also been interested in soft

matter,

electron

crystallography

Materials,

and

the

School

University

of of

Manchester. Xiangli obtained

Ptychographic X-ray tomography as a 3D imaging

her

technique.

BEng

Metrology

Instrumentation and MEng in Materials Science and Engineering. She has registered

Dr Julia Parker

her PhD since 2006 in National University of Julia is an X-ray microscopist at

Singapore. Xiangli has extensive experience and rich

Diamond Light Source, the UK’s

knowledge

national

synchrotron

electron

microscopies,

facility.

microscopies and sample preparation techniques.

Julia is responsible for the

Her current research interest is on minimising

operation of the hard X-ray

focused ion beam (FIB) induced damages and FIB

nanoprobe beamline, supporting

technical development on various types of materials.

nanoscale spectroscopy, diffraction and imaging experiments across the life and physical sciences. Julia joined Diamond in 2007 after completing her PhD at the University of Cambridge. Julia’s current research interests lie in the area of biomineralisation, using synchrotron techniques to unveil details of the structure of calcium carbonates formed by organisms such as shells and studying the crystallisation and formation pathways of calcium carbonates. Ms Jane Woolrich Jane is Materials Engineer and a senior member of the Service Investigation team for Civil Aerospace at Rolls-Royce plc. Her prime role involves the use of both optical and electron microscopy techniques for fractographic analysis, using both established techniques and applying new

ion

ISSUE 65 MARCH 2022

Convergent transmission electron diffraction pattern of Si(111).

Wood sample scanned at 280 nm voxel size - vessels are color-coded to thickness.

TESCAN UniTOM HR The first micro-CT system to provide sub-micron spatial resolution and high temporal resolution dynamic CT in a single, highly versatile system. What can you do with TESCAN UniTOM HR? Contact us today to find out:

www.tescan.com

C O M PA N Y N E W S

Teledyne Photometrics partners with Dotphoton AG

Teledyne Photometrics announces a strategic partnership with image compression solutions provider Dotphoton AG that will enable robust storage of the large amounts of images generated by scientific imaging using integrated combinations of products from the two companies.

of generating 4.9 Gigabytes of data per second. Solutions such as Dotphoton’s Jetraw are necessary for efficient post acquisition data management.” Jetraw’s metrologically accurate raw image compression software reduces image data volume by as much as 10x and is compatible with most visualisation and processing software. Jetraw uses a full information-theoretical model of the image acquisition process, based on the physical properties of both the light and the sensor to ensure optimal performance. Teledyne Photometrics, a part of Teledyne’s Vision Solutions Group, manufactures industry leading scientific CMOS and CCD cameras used in Life Science research. With more than 40 years of experience, Teledyne Photometrics has pushed the boundaries of scientific imaging with the Kinetix, a 10 Megapixel, 95% Quantum Efficient, 500 frame per second sCMOS cameras.

Joe Deasy, Marketing Manager at Teledyne Photometrics notes that this partnership is a move to resolve prohibitive data storage issues caused by the large amounts of data generated during modern scientific imaging. Deasy explains: “Our Kinetix sCMOS high-speed scientific camera is capable

Dotphoton is a Swiss technology company that provides innovative data management solutions for large image data. Dotphoton’s metrologically correct approach to image compression enables file size reduction by a factor of 6-10 while preserving the quality of images and improves the experience of managing large data sets. www.teledyne.com

Contacting the Royal Microscopical Society The offices of the Royal Microscopical Society are at: 37/38 St Clements, Oxford, OX4 1AJ, UK Tel: +44 (0) 1865 254760 For general enquiries email info@rms.org.uk For information about meetings and courses email events@rms.org.uk For membership enquiries email membership@rms.org.uk

www.rms.org.uk

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Olympus Provides Hands-On Non-destructive Testing Instruction to Engineering Students Olympus recently donated OmniScan™ flaw detection equipment to LeTourneau University to support the next generation of non-destructive testing (NDT) inspectors. Olympus phased array ultrasonic testing (PAUT) specialists Curtis Dickinson and Rob Frashefski travelled to the campus in Longview, Texas to offer students inperson instruction. The class they visited, Non-destructive Evaluation and Testing, is a senior level course that teaches engineering students the theory and practice of NDT methods. Using the OmniScan equipment to demonstrate, the Olympus experts discussed PAUT theory and practice with the students in the Welding/Materials Joining Engineering program. “It was a great experience getting to learn about ultrasonic phased array and how to maximise the use of the equipment in our studies,” said Amber Van Duyn, Welding Engineering Junior.

LeTourneau University is a Christian polytechnic university where rigorous engineering is taught through an immersive, hands-on learning approach in a vibrant faith community. The Department of Welding/Materials Joining Engineering is part of the ABET-accredited Bachelor of Science in Engineering (BSE) and Bachelor of Science in Engineering Technology (BSET) degree programs and offers concentrations in Materials Joining Engineering. Olympus is passionate about creating customerdriven solutions for the medical, life sciences, and industrial equipment industries. For more than 100 years, Olympus has focused on making people’s lives healthier, safer and more fulfilling by helping to detect, prevent, and treat disease; furthering scientific research; and ensuring public safety. www.olympus-ims.com

C O M PA N Y N E W S

Linkam works with NIBSC to bring advanced microscopy to lyophilisation Researchers at the UK’s National Institute for Biological Standards and Control (NIBSC), led by Dr Paul Matejtschuk, are using the latest FDM (Freeze Drying Microscope) technology to investigate the development of formulation and freeze-drying processes, with a focus on protein therapeutics. The FDCS196 system for FDM from Linkam Scientific Instruments has been used in the research, and the group uses FDM to establish how different formulations affect the freeze-drying process. The team at NIBSC recently investigated the liposomal freeze-drying process to improve its reproducibility and compatibility with the highthroughput screening of liposomes. Specifically, the study aimed to identify the formulation and process parameters ideal for the freeze-drying of empty and protein loaded liposomes using Ovalbumin (OVA).6 In this research, FDM was used to predict the ideal freeze-drying conditions for liposomecryoprotectant mixtures, by enabling an estimation of the freezing, collapse, and melt temperatures. Results showed that the presence of protein adds stability to both neutral and charged formulations, with the same amount of OVA retained after freeze drying. The study also demonstrated the ability to freeze-dry liposomal formulations in microplates, as well as vials for the rapid screening, preservation,

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and optimisation of liposomal formulations. Liposomal physicochemical characteristics were preserved regardless of the formulation type, with no loss of protein observed. This positions the freeze-drying process outlined in this experiment as an ideal method for screening and improving the longevity potential of pharmaceutical liposomal products. As the biologics market continues to grow, the demand for lyophilised formulations is forecasted to rise. Biopharma companies, for example, require methods to examine each stage and optimise the speed and cost of the process. The availability of accurate FDM methods is therefore vital for the in-depth investigation of freeze-drying processes and structures and is being used to analyse the most challenging formulations, such as liposome drug delivery systems that require sophisticated stabilisation methods. By altering and adapting the three main freeze-drying steps (freezing, primary drying under vacuum and secondary drying through heating) to specific formulations, researchers at NIBSC are providing the tools needed for the pharma industry to improve its drug development processes and maintain drug stability when scaling up to production. www.linkam.co.uk

ZEISS Enters into Strategic Partnership with University of Oxford The Oxford-ZEISS Centre of Excellence (OxfordZEISS-CoE) is being developed in partnership with the Kennedy Institute of Rheumatology (KIR) and the Institute of Developmental and Regenerative Medicine (IDRM) In December 2021, ZEISS entered a strategic partnership with the Kennedy Institute of Rheumatology (KIR) and the Institute of Developmental and Regenerative Medicine (IDRM) developing the Oxford-ZEISS Centre of Excellence (Oxford-ZEISS-CoE). This collaborative partnership promises to deliver a means through which to push the boundaries of both the very latest imaging technologies and analysis approaches, to advance the study of global health and disease. Professor Marco Fritzsche, Scientific Director of the Oxford-ZEISS-CoE said: "We have built a concept that not only gives our researchers access to the latest commercially available optical imaging microscopes, but also to the unique expertise of ZEISS engineers in their Research and Development team. This gives us the freedom to raise questions relating to our areas of biological study that may challenge current

microscopy capabilities, allowing us to continue to develop new technologies and transform microscopy across our research interests." The scope provides a strong grounding for a close working relationship between the development team in the Light Microscopy division of ZEISS and personnel at the University of Oxford. This relationship is already delivering the anticipated impact with instrument modifications being jointly discussed and implemented. One of the disruptive imaging technologies that promises to re-define many experiments at the University is the Lattice Lightsheet 7. The very first UK installation of this system was at the Oxford-ZEISS-CoE in December 2020, and modifications have already been made in order to advance the capability and really push the boundaries of the possible science. Find out what Professor Marco Fritzsche (Scientific Director of the Oxford-ZEISS-CoE) and Dr Helena Corker (Advanced Microscopy Specialist for Lattice Lightsheet technology) have to say about this exciting new collaboration. www.zeiss.ly/AdvancingImagingFrontiers

If you would like your Company News to appear on these pages, please contact infocus Magazine at advertising@infocus.org.uk. The announcements in this Section are compiled by the manufacturers. They in no way represent a recommendation by the Royal Microscopical Society for any particular instrument or equipment.The Royal Microscopical Society does not endorse, support, recommend or verify the information provided on these pages.

NEW PRODUCTS Vision Engineering launches its first 4K resolution digital microscope Vision Engineering Limited (Woking, UK) has announced the launch of it first 4K resolution digital microscope, Makrolite 4K. Makrolite 4K’s superb image quality, 4K resolution and wide dynamic range is suitable for a wide range of complex and high contrast applications.

such as production, lab research, R&D, micro assembly, quality control, inbound/outbound product checking, dissection and re-work. Available in two versions, the console configuration provides direct HDMI connection to the monitor for live display with full control of zoom and all camera settings. Connecting Makrolite 4K to a PC with our dimensioning software, ViPlus, extends its capabilities to include image capture, annotation, on-screen measurement, live overlays, data/image report generation and a range of image processing tools Additionally, the Makrolite 4K solution includes a wide range of stands and objectives, making it a flexible solution equipped to deal with a wide range of demanding inspection tasks.

It provides more fine detail with greater detail shadow and highlight areas, ideal for challenging inspection routines, including reflective subjects, for example solder joints, subjects in shadow, or subjects with low contrast, such as rubber and plastic. Makrolite 4K is flexible, easy to use, and provides high definition video images with a wide dynamic range and up to 330x magnification. It delivers both versatility and high performance in applications

Paul Newbatt, Vision Engineering Group Sales and Marketing Director said: ‘’Makrolite 4K represents a step forward for Vision Engineering in terms of inspection microscope image resolution. It retains the core Vision Engineering values of superb image quality combined with ease of use, and also adds 4K resolution, a choice of console or PC versions, and range of five stands, to deliver a really flexible inspection solution.” www.visioneng.com

Teledyne Photometrics announces the release of its long exposure CMOS camera series Teledyne Photometrics, a division of Teledyne Technologies, announces the release of the Retiga E7—a cost efficient, long exposure optimized CMOS camera boasting low read noise and unprecedented dark current control. The Retiga E7 is a major breakthrough in CMOS thermal noise control for applications that require exposure times stretching from seconds to minutes. Cost efficient cameras that can capture weak signals using long exposures are the foundation of many areas of scientific research, including PCR and protein analysis. Teledyne Photometrics is happy to continue to provide a solution that secures the future of these techniques.

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“The Retiga E7 addresses an urgent issue faced by systems manufacturers in various industries who rely on the low dark current characteristics of CCD devices, many of which will soon be phased out,” notes Joe Deasy, Marketing Manager at Teledyne Photometrics. The Retiga E7 is a 7 Megapixel CMOS camera with low read noise (2.2 electrons) and ultralow dark current (0.02 electrons per second) that is comfortably able to detect weak signals while integrating over several minutes. The Retiga E7 is the next generation of long exposure camera, following the highly successful CCD-based Retiga R-series. www.teledyne.com

New telecentric measurement system delivers TESCAN Announces UniTOM HR-The First Dynamic Micro-CT System to Deliver Sub-micron 3D Imaging for Static Studies and High Temporal Resolution for 4D Time-resolved Studies TESCAN ORSAY HOLDING announces the release of its new UniTOM HR-the first dynamic micro-CT system to offer sub-micron resolution 3D non-destructive imaging for static studies and high temporal resolution for uninterrupted 4D dynamic CT experiments. UniTOM HR is ideal for both industrial and academic researchers that need micro-CT imaging to visualise a sample’s internal structure and also want to gain a deeper understanding of a sample’s behavior under certain environmental conditions. “TESCAN’s dynamic micro-CT portfolio brings fast dynamic CT imaging from the cutting-edge synchrotron to the mainstream laboratory,” states Marijn Boone, product manager, TESCAN. “UniTOM brings together the most sought-after micro-CT capabilities, giving researchers a versatile solution that covers a broad range of 3D imaging and in-situ applications, handles a variety of sample shapes and sizes, and enables 4D time-resolved dynamic experiments.”

UniTOM HR can characterise newly developed materials at the highest possible micro-CT spatial resolution, a requirement for sub-micron scale static 3D imaging. It can also provide researchers with a better understanding of how these new materials, and functional components created from these materials, will behave under changing conditions through real-time, not time-lapse, visualisations.This dynamic capability sets UniTOM HR apart from other microCT instruments on the market. Boone adds: “Many time-dependent processes are unpredictable and capturing the most important aspects of the process may not be possible in an interrupted or timelapse collection scheme. TESCAN has resolved this issue with its dynamic CT technology that collects data throughout the entire process, providing a wealth of information previously unavailable to researchers.” www.tescan.com

Integrated fluorescence light microscope combines light and electron microscopy into one system The Thermo Scientific iFLM Correlative System is the company's first integrated fluorescence light microscope and a value-added component for the Thermo Scientific Aquilos 2 Cryo-FIB. This enhancement enables cell biologists to streamline their sample preparation process for cellular cryoelectron tomography (cryo-ET).

With the iFLM Correlative System, fluorescently labelled areas can be localised in the frozen sample from within the Aquilos 2 Cryo-FIB chamber. This allows users to select specific cellular regions for FIB

milling and facilitates monitoring and validation by helping to ensure the correct target sites are being prepared. This approach reduces the need to use a standalone light microscope to localise areas of interest, avoids transfer steps that bear risks for contaminating samples, and makes the correlation between light and electron microscopy easier and faster than using two separate microscopes. "At Thermo Fisher, we continue to help scientists expand their knowledge of cell biology down to a molecular level using high-resolution cryo-electron microscopy," said Trisha Rice, vice president and general manager of life sciences at Thermo Fisher. "By simplifying the light-to-electron correlation step, and by eliminating a sample transfer step, our new iFLM Correlative System helps users to locate regions of interest faster so that they can gather answers to their biological questions." www.thermofisher.com/aquilos

NEW PRODUCTS Deeper Insights into Material Properties with the In-Situ Lab for ZEISS Field Emission Scanning Electron Microscopes •

Automated in situ workflows for highly reproducible, precise, and reliable operatorindependent data collection • High-throughput data acquisition with highresolution creating statistically representative results • High-quality data for reliable post-processing, such as strain mapping using digital imaging correlation (DIC), powered by GOM • Easy data management Today, ZEISS is introducing its new integrated in situ workflow for ZEISS field emission scanning electron microscopes (FE-SEM). When researchers need to link material performance to microstructure, which is essential for developing novel materials in a highly efficient way, they can now extend their ZEISS FESEM with an in situ solution for heating and tensile experiments. This allows them to observe materials like metals, alloys, polymers, plastics, composites, and ceramics under heat and tension automatically while plotting stress-strain curves on the fly. They can control all system components from a single PC with a unified software environment that enables unattended automated materials testing for up to 24 hours. Core imaging facilities and materials research labs in academia, government and industry will equally benefit from this new solution. In situ materials testing in the SEM delivers

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precise measurement of the dynamic response of microstructures to mechanical load under defined temperature conditions. Thanks to the design of ZEISS Gemini electron optics, the integration of in situ hardware is very straightforward. Materials scientists can easily add information such as local chemical composition or crystallographic orientations using combined analytical techniques (e.g., EDS and EBSD). All ZEISS FE-SEMs are plugged into the ZEISS ZEN core ecosystem, giving users access to ZEN Connect, ZEN Intellesis, and ZEN’s analytical modules, for example. Dr. Michael Albiez, Head of ZEISS Research Microscopy Solutions, comments: “The ability to quantify material microstructure and bulk mechanical properties in a single automated, user-independent experimental environment provides researchers with the tools necessary to design next generation materials for the future low carbon economy.The insitu lab is not only fully integrated but service and application support are also included. What makes our solution unique is that users can define multiple regions of interest (ROIs) and therefore can be sure to never miss interesting areas of their sample.” The solution is available for immediate upgrade on existing ZEISS GeminiSEM 360 & 460 and ZEISS Sigma 500 microscopes or can be purchased with new systems. www.zeiss.com

Linkam announces latest update to its humidity control range of instruments Linkam Scientific Instruments, expert in sample characterisation, has launched the latest update to its range of humidity control systems. The new RHGen Relative Humidity (RH) Controller offers humidity control between 3% and 95% RH, at temperatures from ambient to 85°C, with an upgraded RH sensor and improved connectors, providing environmental control to a variety of Linkam temperature control stages and third-party chambers. Showcasing upgraded internal electronics and a new colour touchscreen, the RHGen allows the user to precisely control the humidity around a sample, without the need for an external dry air supply. Magnetic connectors for the bottle enables easy water changing and refilling for users and when used in combination with a Linkam stage or other sealed chamber device, and the RHGen can offer humidity control between 3% and 95% RH, at temperatures from ambient to 85°C. Accurate humidity control is ensured through the placement of the feedback sensor close to the sample block, and the device can be used in conjunction with light microscopy, Raman, FT-IR and X-ray to offer further sample characterisation. Humidity can have a significant effect on the properties of a material; these include cosmetic surface effects to changes in a sample’s mechanical and electrical properties, and chemical changes such as creating polymorphs. The RHGen brings precise humidity control to a wide range of applications, so that users

from a variety of sectors can understand the impact of humidity on their samples. Examples include: the study of water ingress on internal electrical components in electronic devices including solar cells; examining the effects of humidity, or dry air, on food storage conditions; and understanding the behaviour and breakdown of pharmaceutical compounds to inform packaging decisions, amongst other applications. Duncan Stacey, Director of Sales and Marketing, commented: “Humidity is often overlooked as a testing parameter, as in the past it has been notoriously difficult to control. This device brings essential upgrades to our previous humidity control system, the RH95, which is already widely used by customers who are incorporating environmental control in their research, and the RHGen will provide customers with further accurate and reliable humidity management.” There are RHGen compatible versions of a number of Linkam stages, including the THMS600, LTS420, and Modular Force Stage (MFS), providing an ideal solution for investigating the effects of changing humidity as well as temperature.The device can also be combined with the Inert Gas Regulator (IGR), which acts as a regulated pressure interface between the RHGen and an external gas supply to allow inert gases into the chamber, to replicate unique environmental conditions. www.linkam.co.uke

NuNano launches silicon nitride SENSE probes for atomic force microscopy imaging NuNano’s newest addition of silicon nitride SENSE probes allow AFM imaging of soft samples using contact mode and AC mode in air or liquid. As with all NuNano products, the SENSE probes maintain the company emphasis on guaranteed tip sharpness and minimal variation in mechanical properties. Rigorous inspection of all NuNano’s probes using Scanning Electron Microscopy (SEM) ensures reliable imaging every time. The SENSE probes feature a sharp silicon tip integrated onto a silicon nitride cantilever with gold reflective backside coating. They include the SENSE 20 (0.07 N/m, 20 kHz) and SENSE 70 (0.4 N/m, 70 kHz) probes. These AFM probes are suitable for imaging soft samples due to their sensitivity minimising sample damage. Dr James Vicary, Managing Director at NuNano says: “Despite the disruption over the past two years, our engineering team have pulled out all the stops to bring this new range of probes to market. We’re

really excited to offer AFM users working with soft materials or in liquid environments the benefits that come from our focus on high quality, reliable AFM probes”. NuNano is a UK-based start-up company specialising in the design and manufacture of AFM probes. The introduction of the new SENSE tipped silicon nitride probes adds to NuNano’s existing range of conductive, silicon and tipless silicon nitride probes currently available. www.nunano.com

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NEW PRODUCTS Bringing nanotechnology to the next level: automatic nanoimaging with Park FX40 Atomic Force Microscope To overcome the pitfalls of atomic force microscopy (AFM), Park Systems has made exact science easier than ever. The newly introduced FX40 Automatic AFM expands on the family of researchgrade tools with builtin AI robotics and delivers intelligent scan algorithms for automised nanoscale imaging. Thus, we open the doors to novel nano-range insights for both interdisciplinar y researchers and industry engineers alike. FX40s new optomechanical design yields new levels of data quality and measurement precision with a low noise level that allows to easily perform accurate nano-metrology even on the most challenging samples. Building on the experience of several generations of Park Systems AFMs, the FX40 model achieves a new level of cross-talk elimination. With a

raw out-of-plane motion (OPM) below 2 nm over a scan range of 80 µm before image correction, precise measurements of very large flat samples are readily imaged while avoiding common levelling artifacts. The new FX40 model also excels at imaging delicate samples. Resolving fragile structures with a thickness of just one molecule is the most stringent test of AFM performance. Figure 1 shows AFM phase images of trimesic acid (TMA) monolayers on HOPG acquired by FX40 in ambient conditions. TMA forms a honeycomb molecular network with a thickness of just 3A and a period of 1.7nm. The high-resolution capabilities of the FX40 reveal a number of rotational domains of TMA monolayers as well as some defects. In the full version of this article,we describe the imaging process on FX40 that illustrates the straightforward operation with real-world application examples. By using four different samples, we demonstrate the automation of Park FX40 including easy sample-tosample navigation. www.parksystems.com/fx

Thermo Fisher Scientific Announces New Solution to Enable Whole Virus Research in Higher Biosafety Level Labs Thermo Fisher Scientific unveiled its first dedicated solution for whole virus research—the 60°C heat decontamination solution for its Krios G4 CryoTEM. Cryo-electron microscopy (cryo-EM) has been used to study viral pathogens for more than 20 years, resolving the structures of Zika, Ebola, HIV, and coronaviruses. Despite the achievements of virus research, many pathogens have yet to be studied, and questions about their behaviour remain unanswered. Presently, only few high-end cryo-EM instruments are available in biosafety level 3 labs. This means structural virology is often restricted to isolated proteins, inactivated samples or substituted nonrisk model systems—in other words, those deemed safe for lower biosafety environments. Yet, scientists around the world have an increasing desire to study viral pathogens in their native state. With its new heat decontamination solution for virus research, the Thermo Scientific Krios G4 Cryo-TEM can heat all components within its enclosure to at least 60°C for a prescribed amount of time. The solution also contains components to prevent aerosol release and a second control station to control the microscope from the outside of the biosafety lab.This enables scientists to perform cryo-EM on samples

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confined to higher-level biosafety facilities (e.g., biosafety level 3), and allows Thermo Fisher to offer tailored remote and on-site service solutions. “This is only the start of realising a vision and roadmap to bring the complete in-situ cryo-electron tomography (cryo-ET) workflow to biosafety level 3 facilities,” said Steve Reyntjens, sr. director of product marketing at Thermo Fisher. www.thermofisher.com/cryo-embiosafety

New PRECiV™ Software Enables Microscope Users to Easily Capture Precise, Repetitive 2D Images and Measurements PRECiV™ software enables users in production, quality control and inspection to make precise, repetitive 2D measurements and obtain results that comply with the latest industrial standards. With robust data sharing and security features, PRECiV software makes microscopy workflows faster and more efficient. PRECiV software turns any manual microscope into a comprehensive imaging and measurement platform. The software can control all Olympus conventional industrial microscopes, their coded functions, Olympus motorised nosepieces and Olympus digital microscope cameras. It supports brightfield, darkfield, MIX (directional darkfield), polarization and differential interference contrast (DIC) imaging for flexibility and offers robust colour rendering and high resolution to provide high-clarity images. The software’s intuitive interface is simple to use with a navigation tab that clearly groups the software’s functions—such as observation, acquisition and measurement—using large, clearly labelled buttons. For newer users, advanced settings remain hidden while experienced users can easily access all the available features and functions. The home screen

can be customised so that the features and functions used most often can be readily accessed. PRECiV software enables precise, repetitive twodimensional measurements on a live or recorded image. Powerful functions like auto edge detection to reliably measure the distance between two points, auxiliary lines to make complex geometric measurements and a magic wand for automatic area detection enable inspectors to be confident in their data. Acquiring all-in-focus images that extend beyond the microscope’s field of view is also easy. The extended focal imaging (EFI) function serially acquires multiple images in different focal planes while the panorama function enables users to move the stage across the sample and then stitch those images together into one large image. Using PRECiV software, you can now combine instant EFI and panorama images while keeping both hands on the microscope. For advanced applications like grain sizing or non-metallic inclusions, optional Materials Solutions guide users through the steps required to acquire measurements that comply with the latest standards, including ISO, ASTM and JIS. www.olympus-ims.com

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NEW PRODUCTS Vision Engineering launches VE Cam compact digital microscope Vision Engineering Limited (Woking, UK) has announced the launch of VE Cam, a new, simple to use compact digital microscope for a wide range of applications. Launching at Productronica 2021 in Munich, VE Cam is available in two variants with differing fields of view (FOV). VE Cam 50 (50mm FOV) & VE Cam 80 (80mm FOV) offer the power, speed and efficiency of digital imaging in a compact package. Packed with new and established features, VE Cam enables users to do more with maximum space efficiency and is ideal for many routine inspection tasks. Enhanced productivity features include 10 User programmable presets, six hotkeys for instant one touch access to most commonly used presets and a configurable interface which allows most commonly used user settings to be shown direct on the screen.

Suitable applications include: electronics, mechanical engineering, plastics, additive manufacturing, and ceramics. Paul Newbatt, Vision Engineering Group Sales and Marketing Director said ‘The launch of VE Cam extends the Vision Engineering product to include a compact, simple to use and competitively priced microscope which focuses on delivering an efficient and accurate inspection capability, combined with a mix of designed in productivity features, superb image quality and user control. ‘In addition, both VE Cam 50 and VE Cam 80 utilise digital technology which supports immediate information capture and share across teams, for example WiFi image mirroring which means colleagues and supervisors can view live images without interrupting workflow, thus improving efficiency’. www.visioneng.com

New Copper FIB Lift Out Grids from Agar Scientific Agar Scientific have recently launched new Copper TEM lift-out grids specifically designed for FIB applications.The grids are a secure way to attach TEM lamellas to the posts of the lift-out grid, which then can be imaged easily in the SEM/FIB,TEM or used for EBSD analysis.These are the first of a new range, with more currently in development from Agar Scientific. The FIB lift out grids are high-quality and have crisp, well-defined edges, while remaining competitive in price.

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3-post, comprising of one 50 x 200μm straight post and two 120 x 210μm wide v-notch posts

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3-post with side access, comprising of one 50 x 200μm straight post, two 120 x 210μm wide v-notch posts and the left side removed for better access when using a micromanipulator or nanomanipulator for mounting TEM lamella

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4-post, with two 50 x 200μm straight posts and two 120 x 210μm wide v-notch posts

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5-post, with five 50 x 200μm straight posts

Straight posts can accommodate lamellas on either side of their post.This is the quickest and simplest way to use the FIB lift-out grids, straight posts are ideal for plain-view lamellas or when Energy Dispersive Spectroscopy is required.

Manufactured atAgar Scientific’s dedicated production labs in Stansted, the grids are approximately 35µm (±5μm) thick and the effective grid diameter is 3.05mm. Four different types with either v-notch or straight posts are currently available:

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V-notch posts have a central position that can be used for both small and large lamellas. This is designed for mechanical stability and creating thin lamellas, as both sides of the lamella are attached. All Agar Scientific FIB lift-out grids have thinner edge working areas to ensure better attachment of lamellas and are marked with letters from A to E for referencing. www.agarscientific.com

High Energy Cameras for both Imaging and Spectroscopy applications Raptor Photonics has developed a series of camera platforms for use in the X-Ray detection market, offering either direct or indirect detection. A range of sensors can be incorporated into these platforms to address both imaging and spectroscopy applications, enabling photon detection from VUV (<5eV) through to hard X-Ray (>50keV). Using existing CCD, EMCCD and CMOS core engines, Raptor can now manufacture detection solutions with multiple interfaces to the sampling environment, including: • Feedthrough options for In Vacuum cameras including power, CameraLink, Water and Trigger

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Various entrance windows / ambient light barriers, such as Beryllium or Aluminium • Vacuum or Non-vacuum mounting flanges • Fibre-optic coupled configurations to interface to additional components with a fibre-optic output, such as scintillator plates, streak tubes or MCPs Applications • X-Ray Imaging • X-Ray Diffraction (XRD) • X-Ray Fluorescence (XRF) • X-Ray Plasma Imaging and Diagnostics • Soft X-Ray Microscopy • EUV X-Ray Spectroscopy • X-Ray source characterisation • X-Ray Phase Contrast Imaging • X-Ray Tomography • VUV/EUV/XUV Imaging • Lithography • Crystallography www.qd-uki.co.uk

Digital Surf & Taylor Hobson cooperate to drive roundness metrology forward Digital Surf, global provider of surface analysis software solutions and Taylor Hobson, world leader in surface and form metrology, recently completed a new round of co-developments resulting in the release of an updated version of Metrology 4.0 software with the manufacturer’s brand new Talyrond® 500 PRO, a powerful instrument for roundness metrology. Metrology 4.0 software made its debut in 2018 with the PGI Novus series of surface instruments, and now also allows both measurement and analysis on the Taylor Hobson roundness instrument series. Mountains® platform software features are seamlessly integrated into Metrology 4.0, which allows better control of the measurement process and the direct creation and export of analysis documents. Users working with roundness now benefit from a fluidified and optimised workflow, with the ability to progress from measurement to reporting within the same interface, making the metrology process much simpler and more straightforward. This is particularly useful in a production context (shop floor use). Specific features include high-quality 3D visualisations of cylinders and flatness scans, ease-of-use and desktop publishing allowing users to create personalised analysis documents showing raw measurements and analysis steps performed. Thanks

to the multi-instrument compatibility of Metrology 4.0 software, various measurement types can be represented on the same document, for example, roundness, flatness, cylindricity, surface finish and contour. “Cooperation between Digital Surf and Taylor Hobson goes back almost 30 years” said François Blateyron, director of research & metrology at Digital Surf. “Both teams understand each other. Our development department was able to build on its robust scientific knowledge to adapt to the new challenges of roundness metrology.” “Our combined experience in the field of precision roundness measurement and in-depth analysis software provides unique benefits.” said Jon Gardiner, senior business development manager at Taylor Hobson. “Together we have produced the seamless integration of mechanical hardware and roundness metrology analysis software to deliver a world-class measurement instrument.” www.digitalsurf.com

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NEW PRODUCTS Introducing the premier solution for multiphoton imaging: The Scientifica VistaScope With the largest in-class field of view, Scientifica’s latest addition to their modular portfolio of multiphoton imaging solutions, the VistaScope, is an industry leading development. It will enable researchers to see more cells simultaneously - in fact, twice as many cells - compared to the closest competitor. The monumental Field of View Number of 40 (corresponding to a 2.9mm sample field of view when using a 10x objective) means entire brain regions can be visualised, with a performance close to that of a mesoscope. The even illumination the VistaScope achieves means the entire field of view can be seen clearly, with no darkening towards the edges, facilitating the collection of the highest quality data, from which accurate conclusions can be drawn. Simultaneous imaging and stimulation, independent of wavelength, can be performed using the VistaScope. Made possible by the two identical scan paths, researchers can image and stimulate precise

regions of interest, varying the wavelengths as their experiments require, for more flexible research. Dr Adam Packer, University of Oxford, was an early tester of the VistaScope and found that it “enables high-speed imaging over a very large field of view using commercial microscope objectives.” Dr Packer went on to explain that “the scope is easy to use; it is a straightforward multiphoton scope platform that does not require optical engineering expertise to operate.The fact that it uses standard microscope objectives enables an element of future proofing as multiphoton objective designs continue to be optimised; any new objectives coming out in the coming years should still be usable on this platform.” Lastly, Dr Packer said, “We tested this microscope thoroughly in our laboratory and found it to be industry-leading for large field of view imaging using standardised objectives.” The affordable VistaScope is slimline and compact. Researchers can start with a simple SliceScope upright microscope for widefield fluorescence and electrophysiology, and upgrade by adding a VistaScope scan head as their experiments develop. www.scientifica.uk.com

Nanosurf makes waves with introduction of new AFM imaging mode Nanosurf announces WaveMode, a new AFM imaging mode launched at the 2022 Biophysical Society meeting. WaveMode is the fastest force curve-based imaging mode with application to all samples and all environments and is available exclusively on the DriveAFM. It represents the first commercially available off-resonance mode that can use photothermal actuation of the cantilever – instead of the traditional piezoacoustic actuation – to enable fast, stable, and gentle imaging. This new mode continues the tradition of Nanosurf’s 25-year history of innovation in scanning probe microscopy. WaveMode offers users of all experience levels and backgrounds many advantages, including fast imaging rates, no cantilever tuning, and a fully automated laser and photodetector alignment. This new imaging mode is based on CleanDrive, Nanosurf’s exclusive photothermal actuation of the cantilever; CleanDrive provides stable, low drift, and high signal to noise cantilever tunes that are insensitive to changes in the environment. Providing key advantages in both liquid and air environments, WaveMode enables a faster workflow and improved results for AFM imaging in both the life science and materials science applications.

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The DriveAFM was released in 2020 and is Nanosurf’s flagship AFM platform based on a tip-scanning design combining atomic resolution, imaging capability from the atomic scale through 100 μm, full motorisation, and fast scanning. With innovations in scanner design and optical beam path engineering, the DriveAFM offers superior research performance. Nanosurf was founded in 1997 in Liestal, Switzerland, and has since become one of the most trusted and established AFM brands in the market today. While Nanosurf continues to develop and manufacture AFMs at its Swiss headquarters, it is a global company with direct sales, service and support operations in China, Germany, India, Singapore, the UK and US. www.nanosurf.com

Vision Engineering and ZEISS collaborate to produce DeepFocus 1: an innovative and competitive Extended Depth of Focus (EDoF) microscope solution Vision Engineering, British leading designer and manufacturer of high-quality non-contact measurement, digital 3D visualisation, and ergonomic inspection technologies, is partnering with ZEISS Industrial Microscopy to add an extended depth of focus inspection system to its range of microscopy systems for the first time.

The new DeepFocus 1 system represents a significant collaboration between two of the world’s leading microscope innovators. DeepFocus 1 combines Vision Engineering’s technical and design expertise with ZEISS’ new Visioner 1 long depth of focus digital microscope head.

enables DeepFocus 1 to generate “virtual” lenses with distinctly different curvatures, thus focus planes. This is achieved by changing the orientation of each individual micro-mirror in an orchestrated way. Reshaping the curvature of this “virtual” lens at speed enables ultra-fast focusing and real-time all-in focus imaging and documentation.Three viewing options aid understanding of the subject being inspected. Extended Depth of Focus (EDoF) view shows the top down view of the subject with all details in focus. Height-map view displays height data from the subject aiding visualisation of monochromatic samples and understanding of height. Topographic view shows a simulated 3D visualisation of the subject which can be rotated and manipulated. Both Vision Engineering and ZEISS Industrial Microscopy are well known manufacturers in their respective markets and the collaboration will allow both companies to extend their coverage of the global inspection market.

DeepFocus 1, featuring MALS™ Technology, delivers live, real-time extended depth of focus imaging with depth of focus up to 100x greater than that of a conventional microscope, which removes the need for time consuming post imaging focus stacking by delivering ‘all-in-focus’ images instantly to a depth of up to 69mm.

Paul Newbatt, Vision Engineering’s Group Sales and Marketing Director said “We are delighted to partner with ZEISS in adding DeepFocus 1 to our growing innovative microscopy product range, which further cements our position as a leading microscopy solutions provider in the electronics, and precision engineering sectors”.

Using a micro-mirror array lens system (MALS™)

www.visioneng.com

Olympus scanR High-Content Screening Station v. 3.3 Adds Improved Deep-Learning Capabilities for Fast, Efficient Image Analysis The scanR high-content screening (HCS) station provides fully automated image acquisition and data analysis. Version 3.3 improves the deep-learning technology’s capabilities to reliably separate objects in biological samples using instance segmentation, the ability to detect and delineate distinct objects of interest in an image. Using a self-learning microscopy approach, the scanR system’s AI automatically analyses data in an assaybased workflow. The deep-learning technology can detect cells, nuclei and subcellular objects, and extract features from a list of over 100 object parameters. Version 3.3 significantly improves the deep learning object segmentation capabilities to more accurately segment difficult-to-distinguish objects, such as cells or nuclei that are very close together, like in cell colonies or tissue. In addition to tools to develop neural network models

for specific applications, scanR version 3.3 comes with pretrained neural network models for nuclei and cells.These can be used in a broad range of standard applications, including the ability to distinguish between confluent cells and dense nuclei, eliminating the time to train the neural network. Version 3.3 of the scanR software also includes a well plate calibration assistant that makes it fast and simple to calibrate a new well plate for the system. In addition, a new level of license enables collaborators to open, review and re-gate scanR analysis files for easier results sharing. www.olympus-lifescience.com

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NEW PRODUCTS Updates to Linkam’s CMS196V3n stage further support cryo-correlative fluorescence and electron microscopy With nearly 100 systems proven in the field worldwide, Linkam is the market leader in CryoCLEM. The Linkam CMS196V3n is a cryo-CLEM instrument designed to facilitate the full workflow of CLEM, and recent updates to this instrument allow users to analyse their samples with improved stability and ease.As well as maintaining the sample, it provides proven capabilities of safe handling, making it a simple and reliable process for transferring and imaging cryo samples with an optical microscope, while keeping them free of contamination at all times. With a short start-up time and an optional autofill system providing up to 6 hours of continuous, uninterrupted use, the CMS196V3n is time-efficient and increases workflow productivity. Furthermore, it enables co-ordinate mapping, required to locate the same sample in the fluorescence or electron microscope. The newest updates to this stage include updated hardware and firmware to virtually eliminate vibration and imaging artefacts with improved control of the automated filling sequence, improving the overall stability of the system. The new firmware includes an improved user interface which provides a more intuitive workflow. In order to increase the flexibility of the system, Linkam have also released additional EM grid cassettes. While already supporting the standard planchettes, there is now a new cassette which supports the larger 4.6mm diameter planchette HPF grids.These larger grids are often used for tissue

samples, as well as growing samples directly on the grid. In addition, there are now optional cassettes for both standard EM grids and FEI Autogrids that can be used with high NA and short working distance objectives, increasing the range of objectives that can be used with the CMS196. Both of these new cassettes are compatible with existing CMS196 stages. The CMS196V3 was also used as part of a superresolution (SR) set up. Super-resolution cryo-CLEM was used by researchers at Leiden University Medical Centre (LUMC) on intact mammalian cells, achieving a localisation precision of 27 nm. This was sufficient enough to visualise two microtubule bundles split by intracellular vesicles in the periphery of cells. This is considered to be the first time cryoSR imaging has been performed within intact mammalian cells. www.linkam.co.uk

Laser 2000 presents the NL5 for outstanding deep 3D imaging and long-term live cell imaging A common problem for researchers trying to image live cells is that most of the methods to do so actually damage them, and it is very difficult to image thick samples. If you are trying to obtain 3D images of organoids or embryos this can be very challenging! Laser 2000 is the UK partner of Confocal.nl, who have developed a solution with outstanding performance during deep 3D live cell imaging experiments. The NL5 confocal system is live cell friendly, and presents a high acquisition speed (25 fps), with high resolution and high contrast.

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Higher laser power is harmful for live cells, as it generates phototoxicity and alters the behaviour of the live specimen. When compared to a standard Spinning Disk Confocal microscope, the NL5 required 16x less laser power to obtain a comparable image. In order to demonstrate the capabilities of the NL5 for long-term live cell imaging, a zebrafish embryo was imaged for 24 hours. No signs of phototoxicity were detected and the signal-to-noise ratio was good along the full experiment, not showing any bleaching. The NL5 uses a slit pinhole technology and therefore does not suffer from pinhole cross talk, which occurs when out-of-focus light goes through the surrounding pinholes, limiting the confocality. This effect is more evident when imaging deep within the sample. As this does not occur with the NL5, this makes it the perfect choice for imaging thick specimens. www.laser2000.co.uk/nl5/

PIE Scientific’s Tergeo-EM now with water vapour capabilities Surface treatment using functional OH* groups is important for creating optimal hydrophilic conditions on TEM grids for cryo-EM. PIE Scientific’s Tergeo-EM benchtop plasma cleaner is now available with an easy-to-use and reliable water vapour delivery option that allows water vapour to be used as a process gas for this application. Many surface treatment applications, especially for cryo-EM, need to render surfaces super- hydrophilic. Regular process gasses such as ambient air, argon, oxygen and nitrogen can render specimen surfaces hydrophilic, but they rely on moisture absorbed on the surface of the plasma chamber, specimen surface or as by-products generated by the oxidation of organic materials. When water vapour is dissociated by the plasma, it generates OH* functional groups on the specimen surface rendering it hydrophilic. If the plasma chamber has been plasma cleaned for more than several minutes, the moisture absorbed on the chamber wall and other surfaces is quickly depleted and the density of the OH* functional groups within the plasma chamber will be greatly reduced. To overcome this the water vapor plasma delivery system generates a constant external water vapour source into the plasma chamber. This option is available with new systems and can be retrofitted to the many Tergeo-EMs in the field.

The Tergeo-EM is a fully automatic bench top plasma cleaner specifically designed for TEM and SEM specimen cleaning and glow discharge-type applications. The stainless-steel door accepts two TEM specimen rods from all the current TEM manufacturers - plus an optional adaptor to allow simultaneous cleaning of three specimen rods. The chamber door has two viewing ports to allow process observation and a 20-place holder is available to allow hydrophobic-hydrophilic conversation of TEM grids. Tergeo-EM features “specimen friendly” low intensity pulse mode, which is ideal for TEM applications and has both downstream and immersion plasma cleaning modes. For TEM specimen rod cleaning downstream mode ensures gentle specimen and rod cleaning. Extending the flexibility of the Tergeo-EM, its immersion mode is useful for cleaning SEM parts and processes, such as high-speed etching, ashing and surface modification.Tergeo-EM has unique integrated, patent-pending RF plasma sensing technology, which means the plasma strength is measured quantitatively and displayed in real time. The operator can use the plasma strength data as feedback to adjust gas flow rates and RF power to give desired cleaning speeds – ensuring repeatable and consistent results for users with beginner or expert levels of experience. www.labtech-em.com

If you would like your new product information to appear on these pages, contact infocus Magazine at advertising@infocus.org.uk. The announcements in this Section are compiled by the manufacturers. They in no way represent a recommendation by the Royal Microscopical Society for any particular instrument or equipment. The Royal Microscopical Society does not endorse, support, recommend or verify the information provided on these pages.

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NEWS

Meet the Staff: Chloe Goode

Chloe Goode is Exhibitions Manager and Corporate Member Liaison for the RMS. She has been at the Society for more than 10 years, working closely with exhibitors and corporate partners, and ensuring the smooth running of RMS events. In our latest instalment of ‘Meet the Staff’ we find out more about her role – as well as Chloe’s love of animals and her passion for the great outdoors… Chloe says: “My role is to invite companies to exhibit and sponsor upcoming events, and also looking after our corporate members, dealing with the new member enquiries – and reminding our current members of the benefits of RMS membership. We’re always looking at how we can engage more with our members.” Under normal circumstances (remember those?) much of Chloe’s time is spent liaising with venues and smoothing out the wrinkles ahead of exhibitions, to ensure the best possible networking opportunities for the companies. So how has this changed since Covid and the advent of virtual events?

Working at the RMS Putting on a good event is always a challenge, but what does Chloe find most rewarding about her role, and being part of the RMS team? She says:“It’s when you get good feedback and satisfied exhibitors – when they say they’ve really enjoyed an event, that it gave them good networking opportunities and that they’re looking forward to working with us again.

She says:“One of the big challenges has been trying to work out how we can offer the best possible package to our sponsors in a virtual setting – because we obviously haven’t got the in-person exhibition. That has been difficult.

“A huge part is being a charity and working with the companies, because it is not all about generating sales and they really appreciate that. It’s about the wider community and you are working for the greater good of microscopy.”

“I think initially no one really knew what the best solution was, but as time went on, it became clear that getting sponsors more involved with the conference sessions and scientific programme was the way to go. Having more short talks and workshops in the programme, and less of a focus on the exhibition ‘space’ gives them that guaranteed acknowledgement from the delegates.

Chloe adds: “The RMS brings together new technologies and products with new research – combining the two. In my role you can really see how significant that is. It’s great to be involved with events that promote the use of microscopes –because they are such an important tool in all sorts of research.”

“That took us all a while to realise because I think everyone’s initial instinct was to try to recreate what we had done before, but in a virtual setting – but in practice that is near enough impossible. It made us more appreciative of the online opportunities that we can

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realistically offer sponsors, and helped us to do things better.”

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With 10 years under her belt at the society, it would be interesting to know how Chloe feels things have changed at the RMS – both as a whole and in terms of her role over time. “I would say there’s much more standardisation now, in terms of the way we do things –

things like booking forms for exhibition stands and just generally becoming more efficient and making the most of our online system. We’ve become able to work even more closely as a team, and help each other out because we can see what everyone is doing.” “It has always been a unique place to work, with a small-knit team where everyone helps out and pulls together.”

Early interests Chloe grew up in London, and her early interests included sport and drama. She says: “I liked athletics the 400 metres and the high jump – and swimming. In fact I still enjoy swimming when I get the chance. I also really enjoyed theatre studies and took part in all the school plays.” So what drew Chloe to a career in the events industry? She says: “It was probably because my husband - then my boyfriend - had set up a marquee business with his brother, and the thought was that we could go into it together and I could do the sales / events side of things. But I didn’t want to put all my eggs in one basket so I ended up looking at other options. “After university I did some temping jobs catering at various different events – at places like race courses and private events, and there were always different people on the teams and it was always fun meeting and chatting with people. I knew one lady that had started up her own catering company so I did quite a few jobs with her as well. “I did a three-month events course at the ExCel Centre – coincidentally where mmc used to be held for a time - but I didn’t want to stay in London, so I moved to Oxfordshire and the RMS became my first ‘proper’ job.”

Animals, rally cars and ‘taking it easy’ A love of animals has been a constant factor throughout Chloe’s life, from childhood pets including a rat, guinea pigs and rabbits, to her current, and muchloved dog Nutmeg – a frequent and popular visitor at the RMS offices. Chloe also enjoyed horse riding in her younger years, and even worked for three months at an elephant orphanage in Sri Lanka during a gap-year after leaving school – a job which enabled her to travel around the country at the weekends, taking in the sights, sounds and street food.

Chloe with her family.

Chloe says: “I have always loved the idea of working with animals, as a child I imagined owning a pet shop or becoming a vet. I like to be outside as much as possible – going for dog walks, growing things in the garden. I’m desperate for spring to come so I can get out there and start planting things.” Though she has less time to explore the world these days, with two young children to look after (and number three on the way!), Chloe remains passionate about travelling. Several years ago she visited Thailand with her husband, and impressively, the couple once entered the Mongol rally to raise money for charity. “We drove to Russia in a one-litre Vauxhall Rascal, and we converted the back into a bed”, recalls Chloe. “It took us two weeks to get to Russia – a lot quicker than we thought – but our visas weren’t within the date range they wanted, and there was no flexibility. We couldn’t afford to hang around, so ended up driving back through different parts of Europe. We must have raised around £2,000. I have always loved exploring and camping, and just being on the road and being able to go where you want. When the kids get a bit older we can start doing some more of that again.” In the meantime – and when she gets the chance – you will more than likely find Chloe enjoying a spot of yoga, walking the dog or enjoying an open fire in the evening. “I generally like to take things pretty easy when I can. Especially since the pandemic, I’ve definitely embraced the slow pace of everything.” And there’s absolutely nothing wrong with that! Owen Morton

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Submission Guidelines

infocus is the Royal Microscopical Society’s (RMS) vibrant and striking quarterly magazine for members. It provides a common forum for scientists & technologists who use any form of microscope, including all branches of microscopy. Published four times a year, infocus is free to members of the RMS. infocus features articles on microscopy related topics, techniques and developments, an events calendar, news, event reports, book reviews, new product information, and much more. infocus welcomes submissions of: Articles - Full articles or reviews of general interest to microscopists, of approximately 30004000 words (excluding references), with images/ figures (as many as appropriate, 4-8 as a guide). Longer articles can also be considered. Short Articles - Short topical articles, review articles or articles providing hands-on help for microscopy methods. Primer Articles - Short general articles that are focussed on specific techniques. Debuts - Student articles publishing emerging results from a project. Results may still be incomplete, but areas of progress/problems should be highlighted, with the aim of provoking feedback. Book Reviews – if you are a member of the RMS and are interested in writing book reviews for infocus, please contact Owen Morton owen@rms. org.uk. Please see recent issues of infocus for examples of articles and reviews. To request a sample copy of infocus contact owen@rms.org.uk If you are interested in submitting to infocus, contact: editor@infocus.org.uk

Article Text

• Abbreviations should be used sparingly and only if a lengthy name/expression is repeated throughout the article. When used, the abbreviated name or expression should be cited in full at first usage, followed by the abbreviation in parentheses. • Authors should provide a photograph, brief biography as well as contact information that will be published.

References

References in the text should be in the form Joy (2000) or Joy & Williams (2000). For three or more authors, use the form Echlin et al. (2000). The reference list should: • be listed in alphabetical order of first authors’ surnames. • (where a journal is cited) - include authors’ surnames and initials, date of publication, title of paper, name of journal, volume number, and first and last page numbers. • (where a book is cited) - include authors’ surnames and initials, title of book, year of publication, edition, followed by publisher and town, county/state (and country if necessary) of publication. • (where a URL is cited) – include authors’ surnames and initials, year of publication, title of page, URL and date accessed.

Images / Figures • Figures can be one column/half page width, 65.5 mm or two column/full page width, 135 mm. • Larger images may fill the page/spread.A full page of the magazine is 170 x 250 mm, a double page is 340 x 250 mm. • Text in figures (labels, axis labels, legends, etc) should be Helvetica or Arial, 8pt size. • Figure and table captions should be listed numerically at the end of the article text

• Text should be in a standard font (e.g. Times New Roman or Arial) at a size of 12 pt.

• Line weights and line strokes should have a maximum value of 1 and a minimum of 0.25.

• Articles should begin with a brief summary, which accurately summarises the content and is intelligible without reference to the text.

• For graphs and plots, whenever possible, please submit vectorized images. • As much as possible, please avoid white spaces.

• Footnotes and appendices should not be used unless absolutely necessary.

• All images must be high resolution – 300dpi or more.

• The hierarchy of headings within the text should be clear.

• Submission files should be in CMYK format and can be supplied as tiff, jpeg or eps files.

• Spelling should conform with The Concise Oxford Dictionary and SI units must be used.

• Images MUST include scale bars or field widths where relevant.

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• Total number of images/figures/tables should not exceed 15 including tables.

Proofs

Prior to publication, authors will be sent a PDF of the article by email for approval. Authors should ensure articles are thoroughly checked before submission – proof amendments should be limited to minor corrections only.

One column/half page width, 65.5mm Figure 1. Width of figure or table confined to one column.

Offprints Five hard copies of the issue in which the article is published will be sent to the author, together with an emailed PDF of the article.

Copyright Authors are requested to assign copyright to the RMS. However, authors may make copies of their own articles without seeking permission from the RMS, provided that such copies are for free distribution only (they must not be sold) and provided that infocus is properly acknowledged (issue number, month and page number should be given). Permission to reproduce material from infocus in other publications will not be given to third parties except with the consent of the authors concerned. Authors are responsible for obtaining permission to reproduce copyright material from other sources. Approval for reproduction/modification of any material (including figures and tables) published elsewhere should be obtained by the authors before submission of the manuscript and the source of the material should be properly acknowledged. Authors are responsible for any copyright fee involved. Authors are requested to complete and submit a signed copy of our copyright sign-off form. This is available on the RMS website (www.infocus.org.uk).

Two column/full page width, 135mm

Figure 2. Width of figure or table spanning full width of page.

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11/2/21 9:42 AM

infocus Magazine - Issue 65, March 2022