Zebrafish are rapidly becoming a popular model organism for in vivo studies, particularly for drug screening and toxicology studies. Their benefits include fast development, economical husbandry, and direct amenability to microscopy since embryos are transparent. While imaging is fairly straightforward, in many cases, a substantial bottleneck to automated workflows is image analysis. In this webinar, Dr. Jason Otterstrom and Dr. Alexandra Lubin describe an AI-powered analysis platform developed to enable true high-content screening of zebrafish, and highlight a range of applications where they have validated its performance. In brief, the easy-to-use software automatically identifies the fish outline, and internal anatomy & body regions with no required user inputs. They demonstrate the platform’s applicability in the context of counting GFP-labeled hematopoietic stem cells specifically in the tail region, along with measurement of x-ray induced apoptosis and dual-color analysis. Key Topics Include: - What is high-content imaging and how does it apply to Zebrafish - How Deep-Learning can make analysis of zebrafish images truly high-content by extracting the fish’s anatomy - Learn example assays where automated microscopy can facilitate use of zebrafish for screening studies - One solution to orient zebrafish embryos without manual manipulation through specialized plates and software

1. Alexandra Lubin, PhD
Post-Doctoral Research
Associate
UCL Cancer Institute
Jason Otterstrom, PhD
Application Scientist
IDEA Bio-Medical
Casting a Wider Net in Zebrafish
Screening with Automated
Microscopy and Image Analysis

2. Join Dr. Alexandra Lubin and Dr. Jason
Otterstrom as they discuss the use of deep
learning-powered automated microscopy and
image analysis for fast, in vivo zebrafish
screening.
Casting a Wider Net in Zebrafish
Screening with Automated
Microscopy and Image Analysis

3. Alexandra Lubin, PhD
Post-Doctoral Research Associate
UCL Cancer Institute
PI: Dr Elspeth Payne MBChB PhD
A Versatile, Automated and High-
Throughput Drug Screening
Platform for Zebrafish Embryos
Copyright 2021 A. Lubin, AZoNetwork and InsideScientific. All Rights Reserved.

4. Drug Screening In Zebrafish
• In vivo model – whole living
animals
• Small and transparent
• Relatively high throughput –
produce hundreds of embryos
• Screening embryos – thousands
of compounds could be screened
relatively quickly
By Ed Hendel - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=37054608

5. Imaging Workflow

6. Image Processing

7. Images for Analysis
CD41:GFP Transgenic Fish: Fluorescent HSCs
Z-stack images
Best Z-slice for BF
Max Projection
for Green

8. Problems With Automated Analysis
Fish not in their wells correctly 5-10%
Can we automatically exclude these
from the analysis?

9. Problems With Automated Analysis
Total Fluorescence in ImageJ:
Threshold to remove auto
fluorescence excludes dimmer cells
Adapting Software for Cells:
Picks up areas of fluorescence in
different regions

10. Jason Otterstrom, PhD
Application Scientist
IDEA Bio-Medical
Casting a Wider Net in Zebrafish
Screening with Automated
Microscopy and Image Analysis
Copyright 2021 IDEA Bio-Medical, AZoNetwork and InsideScientific. All Rights Reserved.

11. 1. Zebrafish & IDEA Bio-Medical
2. WiScan® Hermes
3. Deep Learning-powered
image analysis
Summary

12. Post-processing classification &
orientation selection
Multiplexing Fluorescence
& Brightfield
A.I.–driven parameter-
free image analysis
High Content Imaging
Time-lapse & Z-stack
Imaging
Automated, quantitative image
analysis
Automated, AI-powered
Zebrafish Screening

13. 25 years experience in design of
electro-optics and precise motion
systems
Expert scientists in
Biotechnology & Pharmaceutical
sciences
Company

14. X, Y, Z
Motion
10x
 Unique stable-plate design:
Stationary plate, Mobile objective
 Rapid plate scanning
Colors: 4 Fields: 1 50 ms Exposure
Plate Format
Scan Time
(mm:ss)
96 01:39
384 05:25
Round-bottom plates supported!
Figure from: Liu, H. et al. (2016) PLoS ONE 11(10): e0164645
Precision Objective Motion Module

15. X, Y, Z
Motion
 Unique stable-plate design:
Stationary plate, Mobile objective
 Rapid plate scanning
 Automatic 3 objective switching
Precision Objective Motion Module

16. X, Y, Z
Motion
2X 4X 10X 20X 40X 60X
 Unique stable-plate design:
Stationary plate, Mobile objective
 Rapid plate scanning
 Automatic 3 objective switching
 Magnifications 2X – 60X: air, oil, water
Precision Objective Motion Module

17.  Unique stable-plate design:
Stationary plate, Mobile objective
 Rapid plate scanning
 Automatic 3 objective switching
 Magnifications 2X – 60X: air, oil, water
 Up to 7 fluorescence colors: 405nm – 694nm
10x
Fluorescence
Channel
Standard
Configuration
DAPI ✔
CFP
FITC ✔
YFP
TRITC ✔
mCherry
CY5 ✔
Transmission
White LED
✔
Laser Diode ✔
Flexible illumination

18. Microinjection of
bacteria (no label)
GFP-labeled
neutrophils
hh:mm
Flexible
Acquisition –
Time Lapse
4x
Dr. Gillian Tomlinson, UCL

19. 20x Arteries Veins
Flexible
Acquisition –
Z-Stack

20. 10x blood flow
Flexible Acquisition – Video (14-28 fps)

21.  Utilises deep learning to accurately segment
zebrafish in brightfield images.
 Detection of multiple organs and structures.
 Division of fish into anatomical regions.
 Selectively identifies fish in desired orientations.
Attributes Quantified:
Area
Count
Fluorescence intensity
Shape parameters
AI-powered Zebrafish Detection
200 um
Organs: Regions:
E = eye HE = Head Region
O = Otic vesicle Tr = Trunk Region
Y = Yolk sac Ta = Tail Region
B = Bladder
H = Heart
S = Spine
F = Fin

22. Analysis Demo
AI-powered Zebrafish Detection
200 um

23. Alexandra Lubin, PhD
Post-Doctoral Research Associate
UCL Cancer Institute
PI: Dr Elspeth Payne MBChB PhD
Drug Screening:
Targeted Therapeutics
for MDS & AML
Copyright 2021 A. Lubin, AZoNetwork and InsideScientific. All Rights Reserved.

24. Haematopoiesis
Siveen et al. Mol Cancer. 2017 Jan 30;16(1):13.

25. CHIP – Clonal Haematopoiesis of Indeterminate Potential
Steensma DP et al. Blood. 2015; 126:9-16.

26. Dnmt3a as a Target
ZF Dnmt3aa
ZF Dnmt3ab
Human Dnmt3a
Lin, M.-E. et al. 2018. Clinical Epigenetics 10:42.
• Dnmt3a encodes for the protein DNA methyltransferase 3α
• R882H is the most common mutation
• Two Dnmt3a orthologues in zebrafish, Dnmt3aa and Dnmt3ab
• Good conservation between human and zebrafish Dnmt3a –
identical amino acid sequence
• One or other orthologue is expressed in each cell

27. CRISPR Knock-outs
gRNA 4uM
Cas9 mRNA 450 ng/ul
Dnmt3aa
Dnmt3ab
• Guides designed for both orthologues & CRISPR performed by
co-injection with Cas9 mRNA into yolk sack of 1-cell embryos
• F1 stable line generated
• Knock-out mutations for both orthologues
Dnmt3a Mutations In Zebrafish: knock-out

28. Haematopoiesis In Zebrafish
CD41:GFP Transgenic Fish
Fluorescent haematopoietic stem cells (HSCs) in
the caudal hematopoietic tissue – CHT)
• Fast development allows screening at 3dpf
• Transgenic CD41:GFP provides read-out for HSCs
Shi, Xiangguo et al. Blood reviews 30 2 (2016): 119-30.

29. Drug Screening
Mutant
X
Mutant
Stem Cells Depleted
WT
WT
Stem Cells Unaffected
X

30. Imaging Workflow

31. Images for Analysis
CD41:GFP Transgenic Fish: Fluorescent HSCs
Z-stack images
Best Z-slice for BF
Max Projection
for Green

32. Athena Zebrafish Software: Detecting The Fish
Fish
Fin Bladder Yolk
Eye
Brain
Fluorescent
Granules

33. Detecting The Fish
Fish that are not orientated correctly:
• May have a smaller fish area
• May have more than one eye visible
Therefore can define a population of
correctly orientated fish by:
• Fish Is Detected
• Setting a minimum area of the fish
• Only counting wells with exactly one
eye counted
Area Too Small
Two Eyes
No Fish
Correctly Aligned

34. Selected Removed
Identifying Aligned Fish

35. Segmentation of the Embryo
Trunk
Head Tail
Fluorescent Granules
In The Tail

36. Counting Granules In The Tail: Choose Parameters
Analysis Parameters:
Fish : Minimum Area : 950000 mic.
Fish : Maximum Area : 1600000 mic.
Yolk : Minimum Area : 100000 mic.
Yolk : Maximum Area : 300000 mic.
Eye : Minimum Area : 20000 mic.
Eye : Maximum Area : 250000 mic.
Fin : Minimum Area : 15000 mic.
Fin : Maximum Area : 300000 mic.
Granules : Granules Smooth : 3
Granules : Granules Background Subtraction : 15
Granules : Granules Intensity Threshold : 1200
Granules : Granules Maximum Merge Area : 20 mic.
Granules : Granules Minimum Area : 10 mic.
Granules : Granules Maximum Area : 400 mic.
Head : Minimum Area : 15000 mic.
Head : Maximum Area : 1000000 mic.
Trunk : Minimum Area : 25000 mic.
Trunk : Maximum Area : 1300000 mic.
Tail : Minimum Area : 150000 mic.
Tail : Maximum Area : 1000000 mic.
Populations:
On Side : 1<=Count Eye<=1 ; 1<=Count Tail<=1 ;
--Granules > 5 : 6<=Tail:Count Granules<=500 ;

37. Counting Granules In The Tail: Results
Statistics. Average of Tail: Count Granules - On Side
1 2 3 4 5 6 7 8 9 10 11 12
A 38 23 71 50 53 63 55 44 96 84 42 21
B 80 82 77 68 41 48 48 64 75 90 75 46
C 72 62 43 44 57 88 66 67 49 43 43
D 61 48 55 65 54 49 41 67 42 51 64 36
E 43 53 51 40 54 48 39 60 61 46 34 39
F 49 72 66 77 60 57 81 96 71 44
G 54 28 74 55 79 63 61 31 48 82 40
H 22 39 44 53 73 34 71 59 41 48 59 42
Misaligned fish
automatically
excluded

38. Comparing Athena Counts to Manual Counting
0 50 100
0
50
100
Comparison of Athena and Manual
CD41:GFP Cell Counts at 3dpf
Athena Cell Count
Manual
Cell
Count r = 0.8437

39. Athena Counts with Age: 2dpf, 3dpf & 4dpf

40. Testing The Counts: Irradiation
0 Gy
40 Gy
100 Gy

41. Mutants with a known phenotype: Rps14
• Ribosomal protein linked to MDS
• Knock-out line in zebrafish – no
phenotypic difference between
heterozygous and WT embryos
without stress
• Haemolytic stress – phenylhydrazine
(phz)
• Only WT embryos recover

42. Drug Screen Workflow

43. Drug Screening Set-Up
Wide bore tip 200 uL
Zebrafish Embryos 24hpf
PTU-treated & Dechorionated
96-well Plate
8 Embryos per Condition
Plate 1 - WT
Plate 2 - Mutants
Drug Treatment 20 uM
24hpf - 3dpf (48h)
Tocriscreen Library
1120 Biologically Active Compounds

44. Drug Screening
• Use Athena to count HSCs and exclude fish not properly aligned automatically
• Export data and compare drugs to DMSO compounds to look for synthetically lethal compounds:
 Drug reduces cell count compared with DMSO control in double hets but not in WT fish
• Approx 400 compounds tested to date
D
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Dnmt3a CD41:GFP Cell Count 3dpf: Tocris Plate 2 - F7
Cell
Count
ns *
ns
ns
*
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Dnmt3a CD41:GFP Cell Count 3dpf: Tocris Plate 2 - A9
Cell
Count
ns **
*
*
*
Quipazine dimaleate
5-HT3 agonist
C-1
Protein kinase C inhibitor
SKF 91488 dihydrochloride
Histamine N-methyltransferase inhibitor
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Dnmt3a CD41:GFP Cell Count 3dpf: Tocris Plate 2 - B10
Cell
Count
ns *
ns
**
**

45. Other Applications
UCL Cancer Institute
Lubin, A., Otterstrom, J., Hoade, Y., Bjedov, I., Stead, E., Whelan, M., Gestri, G., Paran, Y. & Payne, E. 2020.
A versatile, automated and high-throughput drug screening platform for zebrafish embryos. Biology Open, In Press

46. Double Transgenics: Irradiation
0 Gy
40 Gy
CD41:GFP – HSCs
Lysc:mcherry – myeloid cells

47. Other Applications: Acridine Orange (Irradiation)
0 Gy
40 Gy
Acridine Orange:
a fluorescent apoptosis marker

48. Other Applications: Hair Cell Assay
DMSO
Pentamidine Isethionate
Propantheline Bromide
Chiu L. L., et al. 2008. JARO 9(2): 178-190.
Fluorecent Hair Cell
DNA Marker

49. Other Applications: Angiogenesis
Tran T. C.., et al. 2007. Cancer Research 67: 11386.
Kdrl:mCherry

50. Other Applications: Angiogenesis
DMSO 5uM 10uM 20uM
0
50000
100000
150000
Inhibiting Angiogenesis: SU4312
Total
mCherry
Area
µm
2
✱✱✱✱
✱✱✱✱
✱✱✱✱
ns
ns
ns
DMSO 5uM 10uM 20uM
0
50000
100000
150000
Inhibiting Angiogenesis: AG1478
Total
mCherry
Area
µm
2
ns
✱✱✱
✱✱✱✱
ns
✱✱
ns
Tran T. C.., et al. 2007. Cancer Research 67: 11386.

51. Other Applications: Eye Size
Wycliffe R., et al. 2020. IJDB 65(4-5-6):289-299.
mab +/+ mab +/- mab -/-
50000
60000
70000
80000
90000
100000
Eye Size: mab eye mutants 4dpf
Eye
Size
um
2
ns
*
***

52. Beth Payne
The Payne Lab
UCL Cancer Institute
Jason Otterstrom
The Development Team
IDEA Bio-Medical
Ivana Bjedov
Gaia Gestri
UCL

53. 1. To watch the webinar, go to:
https://insidescientific.com/webinar/casting-a-wider-net-in-zebrafish-
screening-with-automated-microscopy-and-image-analysis/
2. To learn more about the zebrafish in-vivo screening, go to:
https://idea-bio.com/all-applications/application-zebrafish-in-vivo-assays/
Thank You!