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High throughput plant phenotyping for the development of better plants for the future Dirk Vandenhirtz 路 CEO LemnaTec CEO dirk.vandenhirtz@lemnatec.com


The challenges  quantifying human vision  imaging far beyond direct human vision  incorporating human experience of visual evaluation in algorithms  target directed development of solutions  integrating biological systems and technology  customising solutions for specific research needs  keeping systems open for future research developments Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 2


LemnaTec  interdisciplinary team  12 years of experience with image based biological measurement

 in-house development of image processing software  in-house development of hardware  development of comprehensive solutions

 compliance with standards

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 3


Hardware

3D scanalyzer accelerating plant phenomics


Accelerating plant phenomics        

opening new prospects high-throughput screens multiple camera units non-destructive measurement quantitative analysis monitor growth dynamics stress assessment link to genomics

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 5


scanalyzer3D Imaging cabinets Fully automated, comprehensive imaging for plant shoots and roots

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 6


scanalyzer3D multiple imaging 3D imaging of full plants 1.

3.

Speaker: Dirk Vandenhirtz – CEO

2.

1. 2. 3. 4.

top View side View side View 90° more views optional

Date: 11/9/2010

Slide 7


scanalyzer3D scanning modes Scanning in different wavelength and modes

1. infrared light – shoot 2. visible light – shoot

3. near infrared – root 4. near infrared light – shoot 2.

3.

4.

5.

5. fluorescent light – shoot

1. Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 8


scanalyzer3D IR imaging 1.

Scanning – infrared light Quantify temperature differences (e. g. within leaves and between plants)

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 9


scanalyzer3D VIS imaging – shape 2.

Scanning – visible light High-resolution colour images for comprehensive morphological and growth phenotyping

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 10


scanalyzer3D VIS imaging – colour 2.

Scanning – visible light Plant colour classification – Key to plant health, stress, nutrients and senecence

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 11


scanalyzer3D VIS imaging – architecture 2.

Scanning – visible light Plant skeleton analysis – key to growth dynamics, morphology and architecture

   

Speaker: Dirk Vandenhirtz – CEO

separation of stem and leaves information about nodes, length of leaves morphology plant growth phases

Date: 11/9/2010

Slide 12


scanalyzer3D VIS imaging – architecture 2.

Scanning – visible light

   

Speaker: Dirk Vandenhirtz – CEO

separation of stem and leaves information about nodes, length of leaves morphology plant growth phases

Date: 11/9/2010

Slide 13


scanalyzer3D NIR imaging – root 3.

Scanning – near infrared light for roots spatial distribution of water content in soil

0h 2h 4h 6h 8h Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 14


scanalyzer3D NIR imaging – shoot 4.

Scanning – near infrared light (NIR) measuring water distribution and dynamics

0h

4h wheat dried down over 16 h at elevated temperature

8h Speaker: Dirk Vandenhirtz – CEO

16h Date: 11/9/2010

Slide 15


scanalyzer3D flourescence imaging 5.

Scanning – flourescent light blue light (< 500 nm) visualises any related fluorescence

     Speaker: Dirk Vandenhirtz – CEO

Chlorophyll (cont., flash) Green fluo. Protein (GFP) Phenolics Autofluorescence Custom solutions Date: 11/9/2010

Slide 16


Hardware

movingfield linking greenhouse to field


movingfield         Speaker: Dirk Vandenhirtz – CEO

Automating plant handling Plant transport 1-40 kg randomisation Precision watering Multiple solution fertilising Pesticide spraying High density growth Environment monitoring Date: 11/9/2010

Slide 18


movingfield – watering         Speaker: Dirk Vandenhirtz – CEO

individual plant watering plant weighing target humidity watering multiple solution fertilising top or bottom watering salinity control water logging water use efficiency Date: 11/9/2010

Slide 19


movingfield – spraying         Speaker: Dirk Vandenhirtz – CEO

automated spraying closed cabin uniform dosage no greenhouse contamination high quality nozzles spraying at night possible air ventilation active carbon filtering optional Date: 11/9/2010

Slide 20


movingfield – conveyor

 high frequency imaging  changing neighbours Speaker: Dirk Vandenhirtz – CEO

 equilibrating hotspots (light, air humidity) Date: 11/9/2010

Slide 21


movingfield â&#x20AC;&#x201C; conveyor Impressions of the movingfield

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

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movingfield – conveyor  Reliable transport Highly modular  Size adjustable

 Later changes possible/expandable  Plant height up to 3m  Technology taken from automotive + supplier production

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 23


movingfield – conveyor

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 24


movingfield – conveyor 1 kg

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 25


movingfield – conveyor 30 kg

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 26


movingfield – conveyor 30 kg

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 27


Phenotyping global view Customer

Status

Capacity

Sector

ACPFG Adelaide Australia INRA Montpelier France INRA Dijon France IPK I Germany Phytochamber

(up an running) (Finish mid 2010) (Finish end 2010) (up and running)

2400 1400 1482 600

Public Public Public Public

17,93% 10,46% 11,07% 4,48%

IPK II Germany Corn IPK II Germany Wheat Agrobios Itally Keygene Netherlands BASF USA Pioneer / Dupont USA BASF Germany IGER UK Bayer Cropscience Belgium

(Finish mid 2010) (since 2008) (since 2009) (Since 2008) (Since 2006) (Since 2005) (Since 2006) (Finished 2010-2011) (Finished mid 2010)

600 600 500 600 800 1500 300 800 600

Public Public Public Private Private Private Private Public Private

4,48% 4,48% 3,74% 4,48% 5,98% 11,21% 2,24% 5,98% 4,48%

Bayer Cropscience Germany

(Finished end 2010)

1200

Private

8,97%

13382 Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

relative

100,00% Date: 11/9/2010

Slide 28


Phenotyping global view Public

Private

37% 63%

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 29


Phenotyping global view 9000 8000

7000 6000

Planned

5000

Up an running

4000 3000

2000 1000 0

USA

Canada

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Europe

China

India

Australia

South Amerika

Date: 11/9/2010

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Hardware

HTS scanalyzer small plant phenotyping


scanalyzerHTS high resolution imaging of trays and MTPs

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 32


scanalyzerHTS high precision xy table

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 33


scanalyzerHTS special features

Special features  top or backlight  NIR, cloudy day and fluorescent light options

Speaker: Dirk Vandenhirtz – CEO

 trays or multiwell plates  easy exchange of inlays  automatic barcode identification Date: 11/9/2010

Slide 34


scanalyzerHTS 3D laser scanning 3D laser scanning  height of small plants  leaf angle  leaf movement  900 nm laser optional

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 35


scanalyzerHTS 3D laser scanning

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 36


scanalyzerHTS imaging

High-throughput screening  multiple imaging per plate/tray  High precision positioning  6 to 384 well plates  Up to 100 plates per run Speaker: Dirk Vandenhirtz – CEO

 High magnification up to 0.0030 mm / pixel resolution  Up to 384 images per plate (single well mode)  Fast analysis while imaging Date: 11/9/2010

Slide 37


scanalyzerHTS plant assays

 Arabidopsis

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 38


scanalyzerHTS plant assays

 Arabidopsis  identified

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 39


scanalyzerHTS plant assays

 Arabidopsis  identified  analysis

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 40


scanalyzerHTS plant assays

209,68 265,32 205,16 245,08 714,88 747,81 728,6 749,69 1164,8 1231,6 1240,5 1299,2 1843,5 1789,2 1770,6

compactness

478 246 47 124 452 205 606 206 249 123 506 218 153 280 157

com_x

473 199 42 139 467 157 548 128 288 126 500 202 100 272 199

com_y

59,7831 13,95 4,78756 37,5621 37,6127 23,496 58,4241 0,607191 20,0004 21,8416 57,8317 30,1587 16,7452 51,8408 4,32299

extent_x

216924 47568,5 1776 16299,8 176740 28051,8 287785 24207,8 68244 14230,1 225939 41255,4 14998,2 73870,2 24814,3

extent_y

2,08E+09 9,12E+07 212737 1,42E+07 1,51E+09 4,40E+07 2,43E+09 3,92E+07 2,20E+08 1,08E+07 1,95E+09 7,50E+07 1,46E+07 2,41E+08 4,06E+07

com_border_dist

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

7,69E+08 3,50E+07 93493,4 8440890 5,37E+08 1,77E+07 2,30E+09 1,14E+07 1,45E+08 5295860 1,51E+09 5,76E+07 3552560 2,06E+08 1,84E+07

min_rect_area

0,176555 0,216167 0,139705 0,109012 0,196421 0,161805 0,11931 0,22306 0,144451 0,156933 0,110272 0,116942 0,203395 0,102503 0,155294

2nd_mom_pa_abs_lg

2nd_mom_pa_norm_lg

0,065387 0,0829152 0,0613975 0,0649841 0,0698581 0,0651041 0,112856 0,0646321 0,0951499 0,0769298 0,0855621 0,0898901 0,0495543 0,087419 0,0703751

2nd_mom_pa_abs_sm

2nd_mom_pa_norm_sm

id_tag cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped cfu0_cropped

519,701 1048,15 1555,59 2595,89 575,996 1090,66 2069,7 2602,52 599,141 1033,6 2121,24 2557,63 1123,18 2102,49 2634,33

0,741115 0,593351 0,861732 0,875413 0,666077 0,762768 0,664711 0,724342 0,707775 0,703792 0,753275 0,729407 0,769727 0,823688 0,769403

Date: 11/9/2010

Slide 41


scanalyzerHTS movement assays Analysing movement of small organisms  movement quantification  spatial distribution of movements  movement pattern  life/death classification

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 42


scanalyzerHTS feeding assays HT-screening for leaf eating organisms  feeding assays  resistance screens (BT)  organism sizes  mortality assessment

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 43


scanalyzerHTS nematode assays

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Copyright LemnaTec Date: 11/9/2010 Slide2004 44


scanalyzerHTS nematode assays

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 45


scanalyzerHTS nematode assays

 Movement  Shape  Count

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 46


scanalyzerHTS nematode assays Sensitivity of the HTS Scanalyzer at different resolutions 12

movement % area

10 dark green

8

light green 6

yellow orange

4

red

2 0 low resolution Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

high resolution Date: 11/9/2010

Slide 47


scanalyzerHTS movement assays

 Movement  Shape  Count

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 48


scanalyzerHTS cell assays

one image per well of a 96 well plate high depth of field compensate meniscus or layered growth automatic elimination of non-colony objects


scanalyzerHTS cell assays

8h

Resulution 10 Âľm per pixel in the image above Small colony diameter around 20-30 Âľm Higher resolutions available


scanalyzerHTS cell assays


Botrici

scanalyzerHTS fungi assays List of Parameters:  Color of disc  Size of infection  validity criteria dry out

HIT


Erysgt

scanalyzerHTS fungi assays

List of Parameters:  Color of

disc  Size of infection  shape of infection  validity criteria dry out


Hardware

PL scanalyzer low throughput, but a great deal of options


scanalyzerPL        

Speaker: Dirk Vandenhirtz – CEO

top cameras (1 or 2) VIS, Fluo, NIR zoom lens systems backlight, toplight higher plants trays multiwell plates beakers with floating organisms Date: 11/9/2010

Slide 55


Hardware

Cameras Specifications & resolutions


Cameras – Specifications RGB Camera sensor size 2448 x 2050 pixels (h x v pixels)

max. frame rate 17 frames per second (at full resolution) spectral sensitivity 400–700 / 950 nm with / without filter field of application high light conditions excitation/ emission

Speaker: Dirk Vandenhirtz – CEO

Fluorescence Camera 1392 x 1040 pixels 17 frames per second 400–700 / 950 nm low light conditions 420–500 nm / > 510 nm

Date: 11/9/2010

Slide 57


Cameras – Specifications NIR Camera sensor size 640 x 512 pixels (h x v pixels)

max. frame rate 30 frames per second (at full resolution)

IR Camera 320 x 240 pixels up to 40 frames per second

spectral sensitivity 900–1700 nm (NIR) with / without filter

8–14 µm (LWIR)

field of application all light conditions

-30°C to +90°C

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 58


Cameras – Resolutions mm per pixel

pixel per mm

scanalyzerPL Macro (min. zoom)

0.0838

11.936

scanalyzerPL Macro (max. zoom)

0.0149

67.253

scanalyzerHTS Macro (min. zoom)

0.0838

11.936

scanalyzerHTS Macro (max. zoom)

0.0149

67.253

scanalyzerHTS Micro (min. zoom)

0.0167

59.707

scanalyzerHTS Micro (max. zoom)

0.0030

335.342

scanalyzer3D Macro (min. zoom)

0.8379

1.193

scanalyzer3D Macro (max. zoom)

0.1484

6.720

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 59


Spectral sensitivity NIR-Camera

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 60


Software

Software Linking data acquisition with analysis, creating and optimising data flows


Software: architecture

LemnaLauncher LemnaControl

LemnaBase

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

LemnaGrid

LemnaMiner

LemnaShare

LemnaCount

LemnaTrack

Date: 11/9/2010

Slide 62


Software

LemnaLauncher Managing all software processes


Software: LemnaLauncher Main program  DB / Grid / Miner  tree view  snapshot view  reanalyze  database administration  run biotest

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 64


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 65


Software

LemnaControl Monitoring the fully integrated automatisation of all LemnaTec scanalyzer systems


Software: LemnaControl Monitoring of all systems  imaging/image analysis  watering/spraying  randomising plants in the greenhouse/growth chamber  importing metadata from connected databases  linking plants/trays/multiwell plates to specific imaging modes  controlled placing of MTPs on different imaging positions (by robots)  switching lights or heating on and off

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 67


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 68


Software

LemnaGrid Image analyzing software


Software: LemnaGrid Image analysis  Drag and drop modules  High flexibility  Transparent data flow  No programming language necessary  Self assembling image analysis wizard  Exclusive plugin algorithms optional Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 70


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 71


Software: LemnaGrid – Wizard Imaging GridWizard  parameterise each analysis step  direct visual feedback on parameter changes  general explanation of algorithm function  context and grid specific comments on algorithm function Speaker: Dirk Vandenhirtz ��� CEO

Date: 11/9/2010

Slide 72


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 73


Software

LemnaMiner The correlation software


Software: LemnaMiner Querying databases

Speaker: Dirk Vandenhirtz – CEO

easy access to database

storable queries and spreadsheets

fast graphical data survey

complex calculations based on graphical interface

direct link between images and data

transformation of raw data to biological relevant parameters

preorganisation of multi-dimensional data for statistical analysis

data formating for export Date: 11/9/2010

Slide 75


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 76


LemnaMiner – Data analysis grids Data analysis grids  drag and drop modules  high flexibility  Transparent data flow  no database language necessary  complex calculations within snapshots and time series  biological data transformation Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 77


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 78


Software

LemnaBase Handling large datasets


Software: LemnaBase Flexible data base  Linux PostgreSQL or Oracle  up to 100 TB  dynamic structure following result needs  maximum transparency  minimised access time  open interface for LIMS integration Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 80


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 81


Software

LemnaShare Connecting multiple users


Software: LemnaShare Sharing progress  online exchange interface  up- and download of analysis grids  exchange within research projects and academia  exchange between companies and phenotyping facilities  support by LemnaTec  avoiding double development  sharing experience Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 83


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 84


Software

LemnaCount Quantifying biological objects


Software: LemnaCount Reliable count  counting high numbers  assessment of size, colour, shape  object classification  high reproducibility

Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 86


Software

LemnaTrack Movement quantification software


Software: LemnaTrack Quantifying movement  assessment of translational and within organism movement  life/death classification  spatial resolution of movement  movement patterns  Visualising movement Speaker: Dirk Vandenhirtz – CEO

Date: 11/9/2010

Slide 88


Software: LemnaLauncher

Speaker: Dirk Vandenhirtz â&#x20AC;&#x201C; CEO

Date: 11/9/2010

Slide 89


Skeleton Interpretation

Skeleton Interpretation recieving advanced data of plant architecture, plant vectorisation, leaf tracking over time


Image processing approach  the idea is to analyze the basic plant architecture of maize during vegetative growth period  separating stem and leafs  using result of skeleton algorithm to reduce visible shape to a central point chain  achieve stable indexing and counting of leaves  vectorisation of leaves by determing characteristic points inside of each leaf

Speaker: Dirk Vandenhirtz

Date: 11/9/2010

Slide 94


Example of characterisic points

start node highest point

leaf

stem

end point

Speaker: Dirk Vandenhirtz

 The vector information of each leaf is represented by three specific points  The full vector and angle information is avaiable for these three points  The stem itself also contains the nodes and therefore contains additional information about internode distance

Date: 11/9/2010

Slide 95


Leaf tracking over time Each leaf keeps its label inside the plant to follow it individually over time

Speaker: Dirk Vandenhirtz

Date: 11/9/2010

Slide 96


New Parameters Avaiable Parameters for each Leaf are  length from the skeleton algorithm basic point chain  projected leaf area  start node, endpoint and highest point  angles between these points  convex hull from visible shape  all other LemnaTec Parameters for Image Objects

Speaker: Dirk Vandenhirtz

Date: 11/9/2010

Slide 97


Leaf Object Parameters

projected leaf area

Speaker: Dirk Vandenhirtz

convex hull

min. rectangle

Date: 11/9/2010

Slide 98


Data

Leaf Area [pixel]

Leaf Area over Time 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 17.5.09

First Leaf Third Leaf 5th Leaf

22.5.09

Speaker: Dirk Vandenhirtz

27.5.09

1.6.09 Time

6.6.09

11.6.09

16.6.09

Date: 11/9/2010

Slide 99


Data Convex Hull Area over Leaf Angle Convex Hull Area [pixel]

100000 80000 60000 First Leaf

40000

Third Leaf

20000

0 0

Speaker: Dirk Vandenhirtz

0,2

0,4 0,6 Leaf Angle [radian]

0,8

1

Date: 11/9/2010

Slide 100


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LemnaTec Product Presentation