LemnaTec by Dr. Joerg Vandenhirtz

High-throughput Plant Phenotyping

a Boost for Genomics in the 21st Century

Dr. Jrg Vandenhirtz CMO LemnaTec Head of Research and Development [email protected]

LemnaTec

Date: 11/10/2010

Speaker: Dr. Joerg Vandenhirtz CMO Slide 2

founded May 1998 in Aachen, Germany

interdisciplinary team (biology, physics, engineering)

12 years of experience with image based biological measurement

in-house development of image processing software

in-house development of hardware

development of comprehensive and integrated solutions

compliance with international standards

The challenges

Date: 11/10/2010


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

The human eye

How many legs does this elephant have?

The human eye

Count the number of black dots

The human eye

are exactly the same size

The circles in the centers

The human eye

scanalyzer3D accelerating plant phenomics

Hardware

Hardware scanalyzer3D

Accelerating plant phenomics

Date: 11/10/2010


opening new prospects

high-throughput screens

multiple camera units

non-destructive measurement

quantitative analysis

monitor growth dynamics

stress assessment

link to genomics

scanalyzer3D imaging cabinets

Date: 11/10/2010


Fully automated, comprehensive imaging for plant shoots and roots

scanalyzer3D multiple imaging

Date: 11/10/2010


1. top View

2. side View

3. side View 90

4. more views optional

3D imaging of full plants

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3.

scanalyzer3D scanning modes

Date: 11/10/2010


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2. 3.

4. 5.

1. RGB visible light

2. NIR near infrared light

3. FLUOR fluorescent light

4. IR infrared light

5. NIR near infrared root

Scanning in different wavelength and modes

scanalyzer3D VIS imaging shape

Scanning visible light

High-resolution colour images for comprehensive morphological and growth phenotyping

Date: 11/10/2010


1.

scanalyzer3D VIS imaging colour


Plant colour classification Key to plant health, stress, nutrients and senescence

Date: 11/10/2010


infrared light

visible light

near infrared light for roots

near infrared light

fluorescent light

1.


Plant skeleton analysis key to growth dynamics, morphology and architecture

scanalyzer3D VIS imaging architecture

separation of stem and leaves

information about nodes, length of leaves

morphology

plant growth phases

Date: 11/10/2010


1.

scanalyzer3D VIS imaging architecture

Date: 11/10/2010


2.

Scanning visible light Phenotyping of growth under field-density growth conditions

Phenotyping based on complex morphological criteria like: structure orientation, momentum of inertia, height, width, roundness, compactness. 0,00

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axis momentum ratio

bendin

g index

medium heigt, wide,

kinked leaves

high,straight upright

compact straight,

intermediate height

compact curved

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scanalyzer3D NIR imaging shoot

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

Date: 11/10/2010


wheat dried down over 16 h at elevated temperature

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Date: 11/10/2010


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wheat dried down over 16 h at elevated temperature LemnaTec NIR imaging and analysis can cover the whole water dynamics of the drying for wheat LemnaTec NIR cameras are suitable for detection of fine cereals structures of fully grown wheat

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Scanning near infrared light

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Date: 11/10/2010


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Iceberg lettuce highest water content dries out the slowest

Oak leaf lettuce green Oak leaf lettuce red red one dries out faster than green one immediate reactions already in first hour measurable

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scanalyzer3D flourescence imaging

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

chlorophyll (cont., flash)

green fluo. Protein (GFP)

phenolics

autofluorescence

custom solutions

Date: 11/10/2010


3.

scanalyzer3D flourescence imaging

Date: 11/10/2010


Visual and chlorophyl scan of a half boiled leaf

scanalyzer3D IR imaging

Scanning infrared light Quantify temperature differences

(e. g. within leaves and between plants)

Date: 11/10/2010


4.

scanalyzer3D NIR imaging root

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

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Date: 11/10/2010


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scanalyzer3D NIR imaging root

Date: 11/10/2010


Corn plant previously shown was grown in a transparent 8 cm polyacryl column Results of NIR monitoring allow measurement of spatial distribution water content in soil

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drying dynamics based on soil layer

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scanalyzer3D RGB imaging root

Date: 11/10/2010


Static root density profiles growth pattern

Bangui Sweet Corn Prelude Bangui

scanalyzer3D RGB imaging root

Date: 11/10/2010


Dynamic root growth - pattern Day 0 12 25 35 60 100

image and data show the development of roots of one plant in time.

movingfield linking greenhouse to field

Hardware

movingfield

Date: 11/10/2010


automating plant handling

plant transport 1-40 kg

randomisation

precision watering

multiple solution fertilising

pesticide spraying

high density growth

environment monitoring

movingfield watering

Date: 11/10/2010


individual plant watering

plant weighing

target humidity watering

multiple solution fertilising

top or bottom watering

salinity control

water logging

water use efficiency

movingfield spraying

Date: 11/10/2010


automated spraying

closed cabin

uniform dosage

no greenhouse contamination

high quality nozzles

spraying at night possible

air ventilation

active carbon filtering optional

movingfield conveyor

high frequency imaging

changing neighbours

equilibrating hotspots (light, air humidity)

Date: 11/10/2010



Date: 11/10/2010


Impressions of the movingfield


reliable transport (700 $ per plant!)

highly modular

size adjustable (1000)

later changes possible/expandable

plant height up to 3m

technology taken from automotive + supplier production

Date: 11/10/2010


movingfield conveyor 1 kg

Date: 11/10/2010

Speaker: Jrg Vandenhirtz CMO Slide 36


Date: 11/10/2010


Finding the needle in the haystack

Date: 11/10/2010


The LemnaTec Scanalyzer System discovers THE ONE which is relevant

For example, THE plant with 3% increase in value is worth $50m if it obtains 10% market penetration and released 2 years earlier

Automatic screening 10x more efficient than human screening

+3%

Modern breeding or GMO methods are able to produce THOUSANDS of new varieties per week

Phenotyping global view

Date: 11/10/2010


Customer Status Capacity Sector relative

ACPFG Adelaide Australia (up an running) 2400 Public 17,93%

INRA Montpelier France (Finish mid 2010) 1400 Public 10,46%

INRA Dijon France (Finish end 2010) 1482 Public 11,07%

IPK I Germany Phytochamber (up and running) 600 Public 4,48%

IPK II Germany Corn (Finish mid 2010) 600 Public 4,48%

IPK II Germany Wheat (since 2008) 600 Public 4,48%

Agrobios Itally (since 2009) 500 Public 3,74%

Keygene Netherlands (Since 2008) 600 Private 4,48%

BASF USA (Since 2006) 800 Private 5,98%

Pioneer / Dupont USA (Since 2005) 1500 Private 11,21%

BASF Germany (Since 2006) 300 Private 2,24%

IGER UK (Finished 2010-2011) 800 Public 5,98%

Bayer Cropscience Belgium (Finished mid 2010) 600 Private 4,48%

Bayer Cropscience Germany (Finished end 2010) 1200 Private 8,97%

13382 100,00%


Date: 11/10/2010


63%

37%

Public Private


Date: 11/10/2010


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USA Canada Europe China India Australia South

Amerika

Planned

Up an running

Australian Plant Phenomics Facility The Plant Accelerator

Mark Tester

ACPFG

The Plant Accelerator

The Plant AcceleratorTM

High throughput phenotyping of plant populations

4,485 m2 building, 2,340 m2 of greenhouses, 250 m2 for growth chambers

Grow >100,000 plants annually in a range of conditions

4 x 140 m2 fully automated Smarthouses

Plants delivered on 1.2 km of conveyors to five sets of cameras

High capacity state-of-the-art image capture and analysis equipment

Regular, non-destructive measurements of growth, development, physiology

First public sector facility of this type and scale in the world

Owned by University of Adelaide, opened 29 Jan 2010

National facility to support Australian plant research

Full GM and quarantine status

UniSA and ACPFG established a Chair and Assoc Prof in Plant Phenomics and Bioinformatics ($1.5m)

Growth measurements counting pixels

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Berkut Krichauff

meanSE;n=8

Estimation of shoot biomass

The projected shoot area of the RBG images gives a good correlation with shoot biomass

Tested for various plant species wheat, barley

rice

cotton

Arabidopsis

y = 154154x + 19065

R = 0.9205

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5wk old barley plants, 8 cultivars


y = 216363x

R = 0,9493

y = 167807x

R = 0,9649

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But control and salt stressed plants have different area-weight ratios

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Golzarian et al. (2010) IEEE Proceedings Signal Processing, in review


Improved estimate of biomass when age of the plant is taken into account

Y = a0 + a1(G+B+Y)+ a2(G+B+Y)H

(H = number of days after seed preparation date)

(Correction for leaf colour did not greatly improve weight estimates)

(Cross validation run 10x)

scanalyzerHTS

small plant phenotyping

Hardware

Hardware scanalyzerHTS

scanalyzerHTS

Date: 11/10/2010


high resolution imaging of trays and MTPs

scanalyzerHTS

Date: 11/10/2010


high precision xy table

scanalyzerHTS special features

Date: 11/10/2010


Special features

top or backlight

NIR, cloudy day and fluorescent light options

trays or multiwell plates

easy exchange of inlays

automatic barcode identification

scanalyzerHTS imaging

Date: 11/10/2010


High-throughput screening

multiple imaging per plate/tray

high precision positioning

6 to 384 well plates

up to 54 plate per run

high magnification up to 3 m per pixel tech. resolution

up to 96 images per plate (single well mode)

fast analysis while imaging

scanalyzerHTS plant assays

Date: 11/10/2010


Arabidopsis


Date: 11/10/2010


Arabidopsis

identified


Date: 11/10/2010


Arabidopsis

identified

analysis


Date: 11/10/2010


Image processing provides reliable quantitative

data allowing e. g. any kind of statistics and a

calculation of mean values and significances.

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But information describing

growth remains limited.


Date: 11/10/2010


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LemnaTecs image processing allows

highly automated imaging in time series.

But area based growth curves only show

a minor part of the imformation available.

How homogeneous was growth? 0

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Date: 11/10/2010


t=0d t=4d t=7d t=11d

The slope of the curves displaying

growth rates show that most plants

grew at a relatively constant rate but

just started at different sizes e. g.

due to delay in germination.

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Date: 11/10/2010


Morphological assessment - compactness -

compactness is calculated based on the

size independent rotational momentum

of the plant

it describes if the leaves are nearer

around the centroid or farther outside

e. g. showing longer stipes

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Date: 11/10/2010


Morphological assessment - rotational symmetry -

rotational symmetry is calculated based

on the size independent 2nd moment

principal axis ratio

it describes in how far the leaves

alltogether show a symmetric plant

this may lead to completely other

grouping than before-mentioned

compactness

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Date: 11/10/2010


Morphological assessment - excentricty -

while calculated with a different

algorithm excentricity provides here

quite similar grouping results as

rotational symmetry

nevertheless plant A02 shows more

significant distance to A01 and A03

than with rotational symmetry

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Date: 11/10/2010


Morphological assessment - medium leaf width index -

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the medium leaf width index is calculated

from the square length of the plant skeleton

divided by the leaf area

it describes size independent differences in

leaf width integrating stipes, leaves and

overlapping effects.


Date: 11/10/2010


Morphological assessment - area/circumference -

while having some size dependency

left the ratio of leaf area divided by

plant circumfence may allow additional

classification of morphological traits.

as with many parameters each one

provides new aspects for grouping

plants which may be especially suitable

for statistical QTL analysis

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Date: 11/10/2010


Morphological assessment - surface coverage -

surface coverage compares the

measured plant area to the area of a

circle covering the whole plant

this parameter is intended to provide a

calculation how dense the plant covers

the soil in its immediate growth area

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Date: 11/10/2010


Morphological assessment - stockiness -

stockiness is mathematically the

description of roundness.

applied to Arabidopsis images it

separates plants with invisible or

relatively short broad stipes from

plants with long small stipes

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scanalyzerHTS 3D laser scanning

Date: 11/10/2010


3D laser scanning

height of small plants

leaf angle

leaf movement

900 nm laser optional


Date: 11/10/2010


Max field of view 810 mm

Max field of view in height 400 mm

Resolution in height 0.2 mm

Only in combination With HTS Scanalyzer


Date: 11/10/2010


Max field of view 810 mm

Max field of view in height 400 mm

Resolution in height 0.2 mm

scanalyzerHTS movement assays

Date: 11/10/2010


Analysing movement of small organisms

movement quantification

spatial distribution of movements

movement pattern

life/death classification

scanalyzerHTS feeding assays

Date: 11/10/2010


HT-screening for leaf eating organisms

feeding assays

resistance screens (e. g. BT)

organism sizes

mortality assessment

scanalyzerPL

low throughput, but a great deal of options

Hardware

scanalyzerPL

Date: 11/10/2010


top cameras (1 or 2)

VIS, Fluo, NIR

zoom lens systems

backlight, toplight

higher plants

trays

multiwell plates

beakers with floating organisms

Cameras

Specifications & resolutions

Hardware

Cameras Specifications

Date: 11/10/2010


RGB Camera Fluorescence Camera sensor size

(h x v pixels) 2448 x 2050 pixels 1392 x 1040 pixels

max. frame rate (at full resolution)

17 frames per second 17 frames per second

spectral sensitivity with/without filter

400700/950 nm 400700/950 nm

field of application high light conditions low light conditions

excitation/ emission

420500 nm / > 510 nm

Cameras Specifications

Date: 11/10/2010


NIR Camera IR Camera sensor size

(h x v pixels) 640 x 512 pixels 320 x 240 pixels

max. frame rate (at full resolution)

30 frames per second up to 40 frames per second

spectral sensitivity with/without filter

9001700 nm (NIR) 814 m (LWIR)

field of application all light conditions -30C to +90C

Cameras Resolutions

Date: 11/10/2010


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

Spectral sensitivity NIR-Camera

Date: 11/10/2010


Software

Linking data acquisition with analysis, creating and optimising data flows

Software

Software: architecture

Date: 11/10/2010


linking data acquisition with analysis, creating and optimising data flows

LemnaLauncher

LemnaControl LemnaGrid LemnaMiner

LemnaBase LemnaShare LemnaCount LemnaTrack

LemnaLauncher

Managing all software processes

Software

Software: LemnaLauncher

Date: 11/10/2010


Main program

DB / Grid / Miner

tree view

snapshot view

reanalyze

database administration

run biotest

LemnaControl

Monitoring the fully integrated automatisation of all LemnaTec scanalyzer systems

Software

Software: LemnaControl

Date: 11/10/2010


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

LemnaGrid

Image analyzing software

Software

Software: LemnaGrid

Date: 11/10/2010


Image analysis

drag and drop modules

high flexibility

transparent data flow

no programming language necessary

self assembling image analysis wizard

exclusive plugin algorithms optional

Software: LemnaGrid Wizard

Date: 11/10/2010


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

LemnaMiner

The correlation software

Software

Software: LemnaMiner

Date: 11/10/2010


Querying databases

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

LemnaMiner Data analysis grids

Date: 11/10/2010


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

LemnaBase

Handling large datasets

Software

Software: LemnaBase

Date: 11/10/2010


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

LemnaShare

Connecting multiple users

Software

Software: LemnaShare

Date: 11/10/2010

Speaker: Matthias Eberius CMO Slide 100

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

LemnaCount

Quantifying biological objects

Software

Software: LemnaCount

Date: 11/10/2010


Reliable count

counting high numbers

assessment of size, colour, shape

object classification

high reproducibility

LemnaTrack

Movement quantification software

Software

Software: LemnaTrack

Date: 11/10/2010


Quantifying movement

assessment of translational and within organism movement

life/death classification

spatial resolution of movement

movement patterns

visualising movement

References Most Global Players in Breeding and GMO are already using the LemnaTec Technology: BASF Pioneer Dupont Bayer CropScience Monsanto Syngenta Dow Agro Keygene IPK CSIRO ACPFG etc.

Visit our website www.lemnatec.com

Thank you for your attention!

Visit our website www.lemnatec.com

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Documents

LemnaTec by Dr. Joerg Vandenhirtz