High Throughput Phenotpying in Controlled Environments: Matching Design Considerations To Research Requirements
From lab bench to Field Phenotyping
Mark Humble PhenoDays 2012
Slide 2
Mid January 2012 An Evolutionary Process: Developing a Phenotyping System
Original customer requirements within a CER, reducing experimental variability, precision data primarily arabidopsis, camelina, brachypodium, Setaria (viridis) plants in trays and/or small pots (2-4”) ability to scan individual plants plant height would not be expected to exceed 3’, less than 200 mm (8”) diameter is expected parallel experiments in other growth rooms maximize the number of plants we can scan………”primarily interested in forward genetic screens and this would require screening roughly 15,000 plants/screen” , others 2000-4000 imaging: top and side VIS, high res fluorescence, IR, hyperspec space limited
Mark Humble PhenoDays 2012
Slide 4
Key spec’s for designing a HT Phenotyping System
Maximum plant dimensions throughout experiment Floor space and ceiling height available Capacity Maximum pot size and weight Imaging requirements Watering strategies Spraying stations/nutrient stations
Mark Humble PhenoDays 2012
Slide 5
Imaging unit sizes – top+side imaging
Plant sizes
2.5 m
2.0 m
0.5 m
Size of the imaging units mainly depends on max. plant sizes Mark Humble PhenoDays 2012
Slide 6
Plant sizes and imaging units
Size of the imaging units mainly depends on max. plant sizes Mark Humble PhenoDays 2012
Slide 7
Plant sizes – 200mm conveyor
Size of the imaging units mainly depends on max. plant sizes Mark Humble PhenoDays 2012
Slide 8
Plant size on cars – 400 x 400 mm cars On complete belts cars filled with 400*400 mm plants Each 2nd belt left empty and only each 2nd car filled leads to 800*800 mm size of plants
Mark Humble PhenoDays 2012
Slide 9
Scaling up hardware
Growing plant phenotyping systems – adapting to researchers requirements Mark Humble PhenoDays 2012
Slide 10
1st Challenge: floor space availability
Mark Humble PhenoDays 2012
Slide 11
Example of system with growth rooms
Growth rooms of different sizes
Imaging area
Example for a growth room plant phenotyping facility for small plants with diameters lower than 240 mm Mark Humble PhenoDays 2012
Slide 12
Phenotyping system functionality- imaging
Watering
Imaging
Imaging and watering of a plant carrier from a growth room Mark Humble PhenoDays 2012
Slide 13
Additional functionality- action modes
Watering
Randomising Manual Imaging
Example for a growth room plant phenotyping facility for small plants with diameters lower than 240 mm Mark Humble PhenoDays 2012
Slide 14
BUT!- big growth rooms are better
Reducing a growth room to half footprint (50%) can reduce number of plat carriers to a quarter (25%) due to need for aisles etc.
Big growth rooms are better Mark Humble PhenoDays 2012
Slide 15
240 mm Carriers- achieving throughput plant no. top + side imaging
357
714
1071
1428
plant no. only top imaging 1428
3213
Some disadvantages of multiple pots in one tray- watering stress, statistical entity, light conditions, randomisation, weighing
Options for plant increasing plant numbers using trays. Mark Humble PhenoDays 2012
Slide 16
Increasing capacity- 320 plant carriers •Number of plant carriers: 320 (16x20) •External growth chamber dimensions: 6000 mm x 6970 mm •Min. Space needed for conveyor system: 4600 mm x 5570 mm •Full system dimensions: 6000 mm x 11785 mm •Growth cabinets to be removed: 12 •Watering and weighing stations in GC: 1
Mark Humble PhenoDays 2012
Slide 17
Increasing capacity- 432 plant carriers •Number of plant carriers: 432 (16x27) •External growth chamber dimensions: 6000 mm x 8750 mm •Min. Space needed for conveyor system: 4600 mm x 7350 mm •Full system dimensions: 6000 mm x 13550 mm •Growth cabinets to be removed: 14 •Watering and weighing stations in GC: 2
Mark Humble PhenoDays 2012
Slide 18
Increasing capacity- 544 Plant carriers •Number of plant carriers: 544 (16x34) •External growth chamber dimensions: 6000 mm x 10500 mm •Min. Space needed for conveyor system: 4600 mm x 9100 mm •Full system dimensions: 6000 mm x 15300 mm •Growth cabinets to be removed: 16 •Watering and weighing stations in GC: 2
Mark Humble PhenoDays 2012
Slide 19
Increasing capacity- 672 Plant carriers •Number of plant carriers: 672 (16x42) •External growth chamber dimensions: 6000 mm x 12250 mm •Min. Space needed for conveyor system: 4600 mm x 10850mm •Full system dimensions: 6000 mm x 15675 mm •Growth cabinets to be removed: 18 •Watering and weighing stations in GC: 2
Mark Humble PhenoDays 2012
Slide 20
Carrier and plant number survey Number of plant carriers Carriers per belt
320 320 20
432 432 27
544 544 34
672 672 42
Plant number 3 per carrier Plant number 9 per carrier
960 2880
1296 3888
1632 4896
2016 6048
Plant number 16 per carrier External growth chamber dimensions Min. space needed for the conveyor system
5120 6000 mm x 6970 mm 4600 mm x 5570 mm 6000 mm x 11785 mm
6912 6000 mm x 8750 mm 4600 mm x 7350 mm 6000 mm x 13550 mm
8704 6000 mm x 10500 mm 4600 mm x 9100 mm 6000 mm x 15300 mm
10752 6000 mm x 12250 mm 4600 mm x 10850 mm 6000 mm x 17100 mm
12
14
16
18
2
3
3
3
Full system dimensions Growth chambers to be removed Watering and weighing stations total
The results show that 50% more removed growth chambers lead to 110% higher system capacity Mark Humble PhenoDays 2012
Slide 21
Expansion Options
If more growth cabinets are removed now or in future the best rationale would be to start with the biggest possible chamber now on the right and expand later on to the left The system shown here after removal of 24 cabinets would have 816 plant carriers. Expansion options have influence on basic geometry, inversion of order relative to start design. Mark Humble PhenoDays 2012
Slide 22
Mid February 2012
320 Plant carriers – survey concept Max . space for growth chamber plenums
Corridor 1.7 m width double use for system loading and exit Imaging areas with 3 imaging units for small plants
Two doors to growth room needed for access to all parts
Mark Humble PhenoDays 2012
Slide 24
320 plant conveyor- details
watering and weighing station 16 conveyor lanes
20 plant carriers per lane Sliding doors of the growth chamber Mark Humble PhenoDays 2012
•Number of plant carriers: 320 (16x20) •External growth chamber dimensions: 6000 mm x 6970 mm •Min. Space needed for conveyor system: 4600 mm x 5570 mm •Growth cabinets to be removed: 12 •Watering and weighing stations in GC: 1 •Two access doors •One automatic door
Slide 25
Preliminary Costs Provided •Number of plant carriers: 320 (16x20) •External growth chamber dimensions: 6000 mm x 6970 mm •Min. Space needed for conveyor system: 4600 mm x 5570 mm •Full system dimensions: 6000 mm x 11785 mm •Growth cabinets to be removed: 12 •Watering and weighing stations in GC: 1
Mark Humble PhenoDays 2012
Slide 26
March 2012
A dedicated phenotyping space ………we received the results of preliminary costing and are moving toward expanding the Headhouse to the north of our Potting Room for the Phenotyping home……………………. From 6 x 12m to 15m x 9m
Mark Humble PhenoDays 2012
Slide 28
April- June 2012
Customer requirements March 2012 primarily Arabidopsis, camelina, brachypodium, Setaria (viridis) so plants in trays through small pots (3-4”) we would need the ability to scan individual plants plant height would not be expected to exceed 3’, more likely ≤ 2’ less than 200 mm (8”) diameter is expected parallel experiments in other growth rooms maximize the number of plants we can scan………primarily interested in forward genetic screens and this would require screening roughly 15,000 plants/screen Sensors: top and side VIS, high res fluorescence, IR, hyperspec Space limited
Mark Humble PhenoDays 2012
Slide 30
Customer requirements March 2012
primarily Arabidopsis, camelina, brachypodium, Setaria (viridis) so plants in trays through small pots (3-4”) ability to scan individual plants plant height would not be expected to exceed 3’, more likely ≤ 2’ less than 200 mm (8”) diameter is expected Sensors: top and side VIS, high res fluorescence, IR, 240 mm carriers offer most flexibility for pots delivery of up to 4 discrete test solutions (alkali & acidic stressing) “light adaption tunnel”
Mark Humble PhenoDays 2012
Slide 31
LemnaTec design concerns
Delivering quality data- starts with plant imaging Precision watering and test solution delivery in trays Engineering costs tray watering vs single plant watering 120mm conveyor instead of 240mm?
Mark Humble PhenoDays 2012
Slide 32
Separating and imaging individual plants
Plant separation top images is often no problem
But on side images one additional plant is always in the background
While dense growth on the plant carrier is good, for imaging we need separated plants.
Mark Humble PhenoDays 2012
Slide 33
Pot layouts and image numbers Empty plant carrier 240*240 mm
Green circles represent max plant diameter. Black circle represent max pot diameter ad adaptor height
The schemes above show different adaptor plates for different geometries. The numbers or arrows signify how many side images (in addition to the top image) can be made for each of the plants to gain maximum information. Mark Humble PhenoDays 2012
Slide 34
High content phenotyping
Leaf Area [pixel]
Leaf Area over Time 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 17/5/09 22/5/09 27/5/09
Mark Humble PhenoDays 2012
First Leaf
Third Leaf 5th Leaf
1/6/09 Time
6/6/09
11/6/09 16/6/09
Slide 35
High content phenotyping may not be possible
width Angle 6 400 Angle 5 300
wi dt h A ngl e 6
400
hei ght
350 A ngl e 5
300
ar ea t op
height area top
Angle 6 Angle 5
w idth 400 300
height area top
250 200
A ngl e 4
ar ea wi de si de
Angle 4
150
area wide side
200
Angle 4
100
100
Angle 3
50
A ngl e 3
200
ar ea smal l si de
0
area small side
Angle 2
Angle 2
Angle 1 A ngl e 1
Di st 1-2
Di st 5-6
Di st 2-3 Di st 4-5
Di st 3-4
Mark Humble PhenoDays 2012
area small side
0
Dist B-1
Di st B -1
Dist 5-6 Dist 4-5
width Angle 6 400 height Angle 5 area top 300 Angle 4
100 Angle 3
0
A ngl e 2
area w ide side
Dist B-1
200 100
Angle 3
0
Angle 2
area wide‌ area small‌ Dist B-1
Dist 1-2 Dist 2-3 Dist 3-4
Angle 1 Dist 5-6 Dist 4-5
Dist 1-2 Dist 2-3 Dist 3-4
Angle 1 Dist 5-6 Dist 4-5
Dist 1-2 Dist 2-3 Dist 3-4
Slide 36
Smaller conveyor- an option Number of 240mm plant carriers: 320 (16x20)
•Number of 120mm plant carriers: 882 •Individual phenotyping of each plant, watering, weighing, delivery abiotic stressors now possible •Huge increase in system versatility
Mark Humble PhenoDays 2012
Slide 37
Meeting research requirements
primarily Arabidopsis, camelina, brachypodium, Setaria (viridis) plants in trays through small pots (3-4”) ability to scan individual plants plant height & diameter accommodated Sensors: top and side VIS, high res fluorescence, IR, 240 mm carriers offer most flexibility- up to 4 pots for top & side imaging with maximum throughput (significant- capacity 1280) delivery of up to 4 discrete test solutions (alkali & acidic stressing) “light adaption tunnel” Individual watering of pots in trays Maximum platform view for institute benefactors & visitors Future imaging requirements, eg, hyperspec
Mark Humble PhenoDays 2012
Slide 38
LemnaTec/Conviron collaboration Phenotyping conveyor “modules” to fit optimal growth room width Optimised air handling to maximise conveyor foot print including plenum redesign Conveyor design to allow maximum airflow Integrated installation/ shared infrastructure Minimised heat loading Fixed width Conveyor modules transported complete for quick install Custom service solutionseg lighting
Maximised floor space for conveyor by redesigning the AHUs Future imaging space available. Visual appeal is much higher. Mark Humble PhenoDays 2012
Slide 39
Growth room– functional survey Watering/ weighing Conditioning tunnel
Growth area with 1140 plant carriers
Imaging units Imaging area Loading/ unloading area
Growth room Mark Humble PhenoDays 2012
Autom. door
Imaging circle Slide 40
Growth room– watering /weighing Watering/ weighing stations
Growth Area Imaging area
Simultaneous watering and weighing for optimum humidity control and evaporation monitoring Mark Humble PhenoDays 2012
Slide 41
Growth room– randomising Randomising the plants eliminates last bias due to positional environmental effects raising the statistical power of the screening results tremendously
Growth Area Imaging area
System is programmed to allow carrier changes either in full random or in a blocked random design to change row and position in the row systematically Mark Humble PhenoDays 2012
Slide 42
Growth room– imaging & watering Maximising performance by parallel modular use of the phenotyping system
Imaging
Conditioning
Imaging area Watering
Simultaneous watering and weighing in the compartments while one compartment is conditioned, imaged and watered Mark Humble PhenoDays 2012
Slide 43
Meeting research requirements
primarily Arabidopsis, camelina, brachypodium, Setaria (viridis) plants in trays through small pots (3-4”) ability to scan individual plants plant height & diameter accommodated Sensors: top and side VIS, high res fluorescence, IR, delivery of up to 4 discrete test solutions (alkali & acidic stressing) “light adaption tunnel” Individual watering of pots in trays Maximum platform view for institute benefactors & visitors Future imaging requirements, eg, hyperspec If possible, viewing from the GH is required
Mark Humble PhenoDays 2012
Slide 44
Growth room– imaging circle
Conditioning Tunnel
Imaging area
Mark Humble PhenoDays 2012
Obstructed view
Slide 45
Jul- Aug 2012
Conveyor system– 4 x 8 + 6 lanes Key point here is the use of the conveyor modules and addition of a set of additional belts to maximise plant numbers in the growth room (1140). Including the 4 sets of watering and weighing units this makes the concept very efficient to all aspects..
6 + 8 +
8
+ 8 +
Smaller module to adapt to any Conviron growth room length Speaker: Matthias Eberius – CEO
8 =38 lanes =1140 plant carriers
These units can be transported as complete module for efficient setup Date: 10/17/2012
Slide 47
Sep 2012
Meeting Research
primarily Arabidopsis, camelina, brachypodium, Setaria (viridis) plants in trays to small pots (3-4”) ability to scan individual plants plant height & diameter accommodated Sensors: top and side VIS, high res fluorescence, IR, throughput dark adaption Individual watering of pots Maximum platform view for benefactors & visitors Future imaging requirements Delivery of up to 4 discrete test solutions (alkali & acidic stressing)
Mark Humble PhenoDays 2012
Slide 49
Irrigation, nutrient options •LemnaTec systems use own precision pump •Water can be taken from the normal water system or any other source (A) •In all cases self refilling pressureless containers or containers with premixed solutions are used •Each pressureless container can serve two pumps nearby (B) •Water and nutrient solution can be added individually to separate watering and fertilising (C) •Alternatively water and fertilizer can be added proportionally from one container or a proportional valve filling a pressureless container (C)
Watering system are highly customisable Mark Humble PhenoDays 2012
Slide 50
Watering and Test Solution Delivery
Mark Humble PhenoDays 2012
Slide 51
Summary
Design matching research Customer Requirements primarily Arabidopsis, camelina, brachypodium, Setaria (viridis)
Y
Not achieved:
plants in trays to small pots (3-4�) ability to scan individual plants
Y
-throughput of multi1000s
plant height & diameter accommodated
Y
Sensors: top and side VIS, high res fluorescence, IR,
Y
Throughput
Y
dark adaption
Y
Individual watering of pots
Y
Maximum platform view for benefactors & visitors
Y
Future imaging requirements
Y
Delivery of up to 4 discrete test solutions (alkali & acidic stressing)
Y
Mark Humble PhenoDays 2012
Y
-hyperspec imaging
Slide 53
Design matching research HT phenotyping requirements are always varied (application, environment, space availability, budget, throughput) The design process can be lengthy and LemnaTec provides extensive support and options throughout the planning phase The LemnaTec system is very flexible- accommodating various throughputs, pots & trays, imaging technologies, spatial considerations LemnaTec was able to provide options for high throughput phenotyping obstacles but not compromising data quality
Mark Humble PhenoDays 2012
Slide 54
From Start to finish
Mark Humble PhenoDays 2012
Slide 55
LemnaTec Scanalyzer3D Discovery Platform
Mark Humble PhenoDays 2012
Slide 56
LemnaTec Scanalyzer3D Discovery Platform
Mark Humble PhenoDays 2012
Slide 57
THANK YOU!
Mark Humble PhenoDays 2012
Slide 58