ﬂuorescent confocal microscopy system
1 2,3 2,3 1 1 Bertrand Viellerobe , Isabelle Janssens , Karine Gombert , Hedi Gharbi , François Lacombe and Frédéric D 1) Mauna Kea Technologies, 9, rue d’Enghien, 75010 Paris, France 2) CEA, I²BM, Service Hospitalier Frédéric Joliot, 4 place du général Leclerc, 91401 Orsay (France) 3) INSERM U1023, Université Paris Sud, Laboratoire d’Imagerie Moléculaire Expérimentale, 4 place du général Leclerc, 91401 Orsay (France)
Multicolor Probe-based Confocal Laser Endomicroscopy Introduction
In Vivo Molecular Imaging with Cellular Resolution
Today, confocal ﬂuorescence microscopy and mul^photon Macroscopic imaging of Angiostamp® using fDOT/CT microscopy are increasingly used for in vivo studies in The biodistribu^on of Angiostamp was ﬁrst evaluated small animals. Such techniques allow studying the using ﬂuorescence Diﬀuse Op^cal Tomography (fDOT) in structure and the physiology of living organism at cellular nude mouse bearing a subcutaneous xenogran tumor scale. The major limita^ons of such imaging is that 1‐ from NIH/MEN2A cells. This imaging technique has been samples need to be placed conveniently on a conven^onal considerably improved since past decade and allows now microscope stage which require extensive surgical reconstruc^ng and quan^fying ﬂuorescence signal in three prepara^on, and 2‐ rapid image collec^on is required to dimensions inside small animal. fDOT imaging fused with minimize the eﬀects of movement (such as animal X‐Ray Computed Tomography (CT) demonstrated a high breathing). To solve this problem, novel confocal uptake of the tracer in the tumor area. Interes^ngly, the approaches using ﬁber bundle‐based systems have been Mauna Kea Technologies, Paris, France uptake seems heterogeneous in the tumor and seems developed by Mauna Kea Technologies (Paris, France). higher in the booom of the tumor. In subcutaneous Such systems, named Cellvizio®, use extremely small xenogran models, the tumour cannot easily grow to the bundles of ﬁbers, 0.3–2.6 mm in diameter that can contain skin where it cannot ﬁnd a lot of nutrients, but it upwards of 30,000 ﬁbers. Each ﬁber is used for excita^on preferen^ally invades the ^ssue below. The tracer seems delivery and recovery of the emission back through the to have a higher uptake in that zone that should be rich in ﬁber to a detector. Hence, each ﬁber can be compared as new blood vessels. Perfect imaging modality doesn’t exist to date if we consider spatial resolution, sensitivity, ease of use or penetration an independent insect eye. The absolute advantages of capabilities. Conventional microscopy is widely used for in vitro and invasive studies whereas whole body imagers can’t However, although fDOT can now detect ﬂuorescence in this apparatus are size, ﬂexibility, and image collec^on reach cellular details in vivo. the nanomolar range, it has sEll a low (a few mm) spaEal speed (up to of 12 frames/s). Up to now, two Cellvizio® resoluEon that can not permit to have a precise idea of • Conventional microscopy systems were available either with a 488 nm or a 660 nm the biodistribuEon of the probe at the cellular scale. • Requires numbers of animals: andnew ethicsﬁber issues laser beam. substantial Here, we describe the high use costs of a • Allows for In vitro imaging of phenomena at a given time: no dynamic imaging bundle‐based ﬂuorescence imaging prototype (Cellvizio® • Provides high resolution Dual Band) that can perform simultaneous excitaEon • Whole body imagers MRI,nm CT, Optical fluorescence with both lasers (488 and 660 nm) and recovery of • Well suited for in vivo imaging of biodistribution of molecular biomarkers emission signal with two detectors. We validate the • Non invasive: compatible with longitudinal studies system comparing biodistribu^on of a resolution: ﬂuorescent • Cellular information the unreachable due to low spatial RGD‐based probe (Angiostamp®) in diﬀerent region of a CELLVIZIO® DUAL BAND is a multicolor probe-based Confocal Laser Endomicroscope tumor xenogran as well as in diﬀerent organs of a mouse. • Fills the gap between conventional microscopy and whole body imagers (Fig 1 on the right) This ﬂuorescent probe is known to bind the αvβ3 Integrin, • From bench to bedside: Cellvizio® is FDA approved and CE cleared for clinical indications a protein overexpressed at the surface of endothelial cells • Delivers dynamic in vivo fluorescence imaging of molecular events with cellular resolution (1,4 µm) during angiogenesis . • Can access any tissue with minimal invasiveness including deep brain, abdominal cavity, GI tract, etc...
Angiostamp® is surrounding the tumor blood vessels
H. Gharbi*, F. Lacombe
EMIM 2013 | TORINO | ITALY
• Longitudinal studies made possible: evaluation of drug candidates actions on the same animal over time • Turn to Optical Biopsy and real-time diagnostics (Fig 2 below)
Materials and methods • Point Of Care molecular imaging station
Angiostamp® is no
Angiostamp® is not surrounding the blood vessels of muscle FITC-dextran
Angiostamp® is not accumulated in liver FITC-dextran
Angiostamp®is not accumulated in spleen FITC-dextran
Fig 1 Cellvizio bridges the gap between conventional miicroscopy and whole body imagers
Fig. 1: BiodistribuEon of Angiostamp® analyzed by fDOT/CT imaging ● Ethics Statement Fluorescence signal reconstructed in 3D (colored) was fused to CT All animal use procedures were in strict accordance with the imaging of the mouse (gray). recommenda^ons of the European Community (86/609/CEE) and the French Na^onal Commioee (décret 87/848) for the care and use Microscopic imaging of Angiostamp® using Cellvizio® of laboratory animals. ● Animal model Dual Band Female nude mice (~23 g) were subcutaneously injected with 106 Following fDOT imaging, the mice were injected with FITC‐ Angiostamp® is eliminated by glomerulus of kidney Angiostamp® Fig 2 Optical biopsy avoids C634Y tumor cells NIH‐MEN2A expressing the oncogen RET . Aner 15 tissue samples with by providing works with a large variety of fiber-optic probes that have been designed to fit with Dextran before imaging the ﬁber bundle‐based Cellvizio® Dual Band FITC-dextran AngioStamp ® Merge FITC-dextran days, mice have a tumor (~30‐50 mm3). dynamic in vivo microscopic various applications constraints, with diameters as small as a needle tip (300 µm) or with resolution that ﬂuorescence imaging prototype (Cellvizio® Dual Band). ● In vivo ﬂuorescence imaging using fDOT/CT images in a non orMicroscopy minimally Improvement of Fibered Fluorescence images of can reach 1,4 µm. The system is able to simultaneously track two diﬀerent molecular signatures in vivo and The instrument allowed to acquired in real‐^me image of invasive manner. Angiostamp (10 nmol) was intravenously injected into the tail of in situ, allowing therefore colocalization studies to be conducted on the go in the living animal. The individual cells in the brain of live mice Improvement of Fibered Fluorescence Microscopy images of e a l t i m ewith s t r u FITC‐Dextran cture and anesthe^zed animals. 3D ﬂuorescence images were acquired 3h or blood vessels Rlabeled and the signal system’s wavelengths (488 and 660 nm) cover a large spectrum of in vivo compatible fluorescent dyes, function characterization and individual in the brain antibodies of live mice Jesus Pascual-Brazo, Veerle Reumers, Sarah-Ann Aelvoet, Zeger Debyser, Veerle Baekelandt cells proteins, biosensors, or genetically engineered animal models used routinely in translational 7h post‐injec^on using a prototype op^cal imager (TomoFluo3D). CT from Angiostamp®. Thanks to the high ﬂexibility of the physiopathology diagnosticsFaculty is Pascual-Brazo, for Neurobiology and Gene Therapy. Department of Neurosciences. of Medicine,Veerle K.U. Reumers, Leuven Jesus Sarah-Ann Zeger Debyser, Veerle research. The Aelvoet, tremendous advances in Baekelandt biomarker discovery is putting in vivo diagnostics to a whole new imaging was perform using the SkyScan 1178 Laboratory high‐throughput system diﬀerent organs can easily been analyzed as well as Laboratory for Neurobiology and Gene Therapy. Department of Neurosciences. Faculty of Medicine, K.U. Leuven therefore possible. precision level. Cellvizio® Dual Band sets the stage to a better understanding of molecular pathways that micro‐CT (Skyscan, Kon^ch, Belgium). Fusion of fDOT with CT was diﬀerent part of the tumor xenogran (scheme 2). are leading to cancer, inflammation, infection or neurodegenerative diseases. performed using the Brainvisa medical imaging processing sonware INTRODUCTION MITOCHONDRIAL REDOX STATE IN LIVE MICE INTRODUCTION MITOCHONDRIAL REDOX STATE IN LIVE MICE (hop://brainvisa.info/index_f.html) . Imaging techniques, such as magnetic resonance imaging and Imaging techniques, such as magnetic resonance imaging and ● In vivo ﬂuorescence imaging using Cellvizio® prototype A about B C In vivo biodistribution and kidney clearance of αvβ3 positron emissionMicroprobe tomography, have provided huge information positron emission tomography, have provided huge information about UltraMiniO Using the endoscopic system, we demonstrated that we can simultaneously observe th Aner fDOT imaging , 1mg the of structure FITC‐dextran (500 kDa) the structure and function of the brain during the last years but the low integrin molecular marker and function of the brainwas during the last years but the low Resolution 1,4 µm resolution and acquisition times limits the information that can be intravenously injected in resolution animals before surgery. Then, the information that can be blood vessels. We observed a high accumula^on of Angiostamp® surounding blood v and acquisition times limits Working 60 µm obtained with Distance these techniques. obtained withwas theseperformed techniques. with the Animal model fiber-optics bundle Fluorescence imaging at the cellular level A 30000 new technology developed by MaunaKea®, called Fibered Angiostamp® was localised close to blood vessels of healthy ^ssue such as muscle, s A new technology developed by MaunaKea®, called Fibered Fluorescence Microscopy, is trying to fill the gap between the existing Female nude mouse bearing MDA MB231 tumor ﬁbered confocal microscope Cellvizio® Dual Band from Mauna Kea brain imaging techniques. The Cellvizio microscope, based in a fiber Cellvizio® allows us to conﬁrm that the macroscopic image obtain by fDOT correspon Fluorescence Microscopy, is trying to fill the gap between the existing 50 µm 50 µm 50 µm xenograft underwent intravenous injection of 1 mg optic probe that transport the emission and fluorescent light to the Technologies. The device consists in a ﬂexible sub‐millimetric brain imaging techniques. The Cellvizio microscope, based in a fiber Hindlimb Hindlimb Tumor FITC-Dextran 500 kDa (Sigma-Aldrich) andthe 10 nmol maybe also to uptake by tumor associated macrophages expressing αvβ3 Integ scanning unit, is able to acquire confocal images with cellular microprobe containing thousands op^cal that light and fluorescent light to the opticof probe thatﬁbers transport thecarry emission Redox imaging. Lentiviral vector targeting redox sensitive protein (roGFP) to Angiostamp® 700 (Raft RGD, fluOptics) resolution (3 μm axial resolution) of deep brain regions in live animals. monitoring of two ﬂuorescent signals by endomicroscopy can be useful to v the mitochondria was designed and produced. Image acquisition revealed scanning unit, is and able660 to nm acquire confocal images with cellular However, it is necessary to introduce a fluorescent dye or protein to from two con^nuous laser source at 488 nm to the redox to spikes of 660 Individual 488 nm(roGFP) nm cells in the hippocampus Mergeof live mice. ImageCell® Redox the imaging. Lentiviral vector targeting redox sensitive protein visualize cells, which usually generates background during the resolution (3 μm axial resolution) of deep brain regions in live animals. D macroscopic imaging and it opens a new avenue to monitor in vivo molecular events software was used to record images at a frequency of 12 Hz, for quantification living ^ssue. The ﬂuorescence emioed aner excita^on by the imaging process. Optimization of viral vector technology accordingly the mitochondria was designed and produced. Image acquisition revealed However, it is necessary to introduce a fluorescent dye or protein to and representation of the intensity of the fluorescent signal. Raw data of A, B | Optical biopsy of hindlimb vessels. with the characteristics of the technique can improve the quality of the redox spikes of Individual cells in the hippocampus of live mice. ImageCell® ﬂuorophores staining the ^ssue species is sent back to the signal intensity was plotted for every time point. visualize the cells, which usually generates background during the images acquired Endothelial wall cells visible as well as blood flow software was with usedthis to microscope. record images at a frequency of 12 Hz, for quantification apparatus, where a dedicated set of algorithms reconstructs images imaging process. Optimization of viral vector technology accordingly and representation of the intensity of the fluorescent signal. Raw data of C | Tumor vessels and tumor associated with the characteristics of the technique can improve the quality of the in real ^me at a frame rate of 12 frames per second. The probe that signal intensity was plotted for every time point. macrophages mixed with endothelial cells FIBERED FLUORESCENCE MICROSCOPY CALCIUM IMAGING OF SEVERAL CELLS IN LIVE MICE images acquired with this microscope. was used is a UltraMiniO probe with 30,000 op^cal ﬁbers, a Cellvizio ® Dual Band is a confocal microscope which makes D | Optical slicing of the kidney, exhibiting  Garanger, E., Boturyn, D., Jin, Z., Dumy,50P.,µm Favrot, M.C. and Coll, J.L. (2005) The authors would like to th use of a 488 or 660 nm excitation which is injected one by AngioStamp® elimination beside vessels. GCAMP3 IMAGING 50 µm 50 µm IN THE HIPPOCAMPUS 240x240 µm ﬁeld of view, and a 1.4 µm lateral resolu^on.
IN VIVO OUTCOMES
MOLECULAR VIROLOGY & GENE THERAPY LEUVEN VIRAL VECTOR CORE - LVVC
NEUROBIOLOGY & GENE THERAPY
MOLECULAR VIROLOGY & GENE THERAPY LEUVEN VIRAL VECTOR CORE - LVVC
NEUROBIOLOGY & GENE THERAPY
PRINCIPLE & ARCHITECTURE
New multifunctional molecular conjugate vector for targeting, imaging, and assistance for fDOT/CT ima one in tens of thousands of tiny fibers optics grouped in a Kidney vasculature AngioStamp® clearance Overlay of the two FIBERED FLUORESCENCE MICROSCOPY CALCIUM IMAGING OF SEVERAL CELLS FITC IN LIVE MICEtherapy tumors. Mol Ther, channels 12, 1168-1175. “Agence Na^onale pour la R Dextran in theof glomerulus Fiber bundle flexible fiber bundle. Excitation is conducted by the fibers European Molecular Ima down to the tissue to be examined whereGCAMP3 it is focused by Dynamic mouse colon imaging and macrophages IMAGING IN THE HIPPOCAMPUS  Garofalakis, A., Dubois, A., Kuhnast, B., Dupont, D.M., Janssens, I., LSH‐2004‐503569]. Microscope description. system is composed of 2 main parts: laser some distal optics which defines the field of view,The the lateral 488 nm 660 nm Merge Scheme 2: IllustraEon of diﬀerent part of the tumor that can be targetting during Mackiewicz, N., Dolle, F., Tavitian, B. and Duconge, F. (2010) In inflammation vivo scanning unit and the fiber optic probe. The light from a photodiode and the axial resolution of the system. Endogenous or imaged by the Cellvizio® Dual Band laser is injected in every microfiber optic of the probe, which transports validation of free-space fluorescence tomography using nuclear imaging. Opt exogenous fluorescence is thenthe produced, Animal model light till thewhich tissue. is Thecollected emitted light is transported by the same 35, of3024-3026. CalciumLett, Imaging several cells in the hippocampus of live mice. Calcium microfiber the detector. towards The S-300 probe used for these experiments by the very same individual fiber and tillredirected a Balb/c mouse colon inflammation model sensitive protein GCaMP3 was expressed employing AAV vectors. ImageCell® contains 10.000 microfibers. software was used to record images at a frequency of 40 Hz, Topical spray of Acryflavine to reveal cypts singleof detector, an laser avalanche photodiode (APD). Scanning Microscope description. The system is composed 2 main parts: Procedure. Animals were anaesthetized by intra-peritoneal injection of the laser the proximal ketamine/medetomidine end of the fiber and bundle scanning unit and the fiber optic probe. The light fromonto a photodiode placed inisa stereotactic device. The probe GCAMP3 IMAGING IN THE OLFACTORY BULBstructure Please contact: francois 0,5 mg AminoSPARK (Perkin Elmer) nIR was slowly introduced in the target brain area and images acquired at laser is injected in every microfiber optic of the probe, which performed by atransports combination of a two fast oscillating mirrors, 50 µm 50 µm 50 µm fluorescent nanoparticle intravenous or frede the light till the tissue. The emitted light is providing transported the same anby overall frame rate 12Hz of 9 frequency, to 50 frames per second Image processing. ImageCell® to select regions of mice. Calcium Calcium Imaging of severalsoftware cells inwas theused hippocampus of live Distal optics administration (tail vein) microfiber till the detector. The S-300 probe used for these experiments Colon crypts Macrophages Macrophages (fps), which compensates for interest, motion artifacts. to protein quantify the Dedicated intensity of the fluorescent signal and to sensitive GCaMP3 was expressed employing AAV vectors. ImageCell® Real Time Acryflavine AminoSPARK 680 distribution contains 10.000 microfibers. Vessels are visible by negative contrast represent the data. Raw datatoof images signal intensity was plotted forHz, every image processing then operates in real time first software was used to record at a frequency of 40 Image Processing time point. Procedure. Animals were anaesthetized by intra-peritoneal injection of compensate for fiber-to-fiber diﬀerences in transmission and Tissue ketamine/medetomidine and placed in a stereotactic device. The probe GCAMP3 IMAGING IN THE OLFACTORY BULB Scheme 1: Cellvizio® Dual Band system background, but also to remove the well-known INCREASED SIGNAL/NOISEfiber RATIO AFTER OPTIMIZATION was slowly introduced in the target brain area and images acquired at In vivo quantification of Calcium spikes in honeycomb pattern and reconstruct a smooth and readable 12Hz frequency, olfactory bulb neurons using GCaMP3 Image processing. ImageCell® software wasimage. used to select regions of
interest, to quantify the intensity of the fluorescent signal and to represent the data. Raw data of signal intensity was plotted for every time point.
INCREASED SIGNAL/NOISE RATIO AFTER OPTIMIZATION
Animal model Balb/c mouse, GCaMP3 loaded AAV local transfection. HIPPOCAMPUS HIGH TITERS HIPPOCAMPUS LOW TITERS HIPPOCAMPUS OPTIMIZED A 300 µm bevelled probe is inserted into the Conventional and optimized viral vectors were stereotactically injected with In vivo neural activation Calcium spikes Calcium Imaging of Quantification several cells inofthe olfactory bulb of live mice. Calcium bulb under a stereotaxic frame viral vectors engineered to express GFP in the hippocampus. Comparison olfactory in the olfactory bulb into Regions of interest sensitive protein GCaMP3 was expressed employing AAV vectors. Sequential of the signal/noise ratio after conventional (high and low titers) and optimized viral vectors transduction was carried out.
activation of neighboring cells was plotted at frequency of 12Hz.
C O N C L U S I O N S • Optimized viral vector technology increased the signal/noise ratio of Fibered Fluorescence Microscopy images in the hippocampus of live mice.
• GCaMP3 allows to record calcium levels of several cells in live mice using this new technique. HIPPOCAMPUS HIGH TITERS
HIPPOCAMPUS LOW TITERS
Conventional and optimized viral vectors were stereotactically injected with viral vectors engineered to express GFP in the hippocampus. Comparison of the signal/noise ratio after conventional (high and low titers) and optimized viral vectors transduction was carried out.
• Mitochondrial redox state can be monitored in vivo using roGFP in vivo with cellular resolution.
1- Vercauteren et al., Multicolor pCLE, SPIE Bios 2013, Calcium Imaging of several cells in the olfactory bulb of live mice. Calcium sensitive protein GCaMP3 waswasexpressed employing AAV vectors. Sequential ACKNOWLEDGEMENTS. A plasmid for mito-roGFP provided by S.J. Remington (University of2Oregon,USA). GCaMP3 from Addgene (L. Looger).of This work has been supported by IWT-SBO/060838 Brainstim, Brazo et was al.,obtained Improvement Fibered Fluorescence Microscopy SCIL programme financing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects. activation of neighboring cells was plotted at frequency of 12Hz.
C O N C L U S I O N S
individual cells in the brain of live mice, WMIC 2012 3- Ducongé et al, Simultaneous imaging of two diﬀerent signals using a new fibered confocal microscopy system, WMIC 2011
• Optimized viral vector technology increased the signal/noise ratio of Fibered Fluorescence Microscopy images in the hippocampus of live mice. • GCaMP3 allows to record calcium levels of several cells in live mice using this new technique. • Mitochondrial redox state can be monitored in vivo using roGFP in vivo with cellular resolution.
ACKNOWLEDGEMENTS. A plasmid for mito-roGFP was provided by S.J. Remington (University of Oregon,USA). GCaMP3 was obtained from Addgene (L. Looger). This work has been supported by IWT-SBO/060838 Brainstim, SCIL programme financing PF/10/019 and IWT-O&O JANSSEN-DEPVEGF projects.
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