Automated, high-throughput quantification of EGFP-expressing neutrophils in zebrafish by machine learning and a highly-parallelized microscope.
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2023
2023
Historique:
received:
07
09
2023
accepted:
26
11
2023
medline:
7
12
2023
pubmed:
7
12
2023
entrez:
7
12
2023
Statut:
epublish
Résumé
Normal development of the immune system is essential for overall health and disease resistance. Bony fish, such as the zebrafish (Danio rerio), possess all the major immune cell lineages as mammals and can be employed to model human host response to immune challenge. Zebrafish neutrophils, for example, are present in the transparent larvae as early as 48 hours post fertilization and have been examined in numerous infection and immunotoxicology reports. One significant advantage of the zebrafish model is the ability to affordably generate high numbers of individual larvae that can be arrayed in multi-well plates for high throughput genetic and chemical exposure screens. However, traditional workflows for imaging individual larvae have been limited to low-throughput studies using traditional microscopes and manual analyses. Using a newly developed, parallelized microscope, the Multi-Camera Array Microscope (MCAM™), we have optimized a rapid, high-resolution algorithmic method to count fluorescently labeled cells in zebrafish larvae in vivo. Using transgenic zebrafish larvae, in which neutrophils express EGFP, we captured 18 gigapixels of images across a full 96-well plate, in 75 seconds, and processed the resulting datastream, counting individual fluorescent neutrophils in all individual larvae in 5 minutes. This automation is facilitated by a machine learning segmentation algorithm that defines the most in-focus view of each larva in each well after which pixel intensity thresholding and blob detection are employed to locate and count fluorescent cells. We validated this method by comparing algorithmic neutrophil counts to manual counts in larvae subjected to changes in neutrophil numbers, demonstrating the utility of this approach for high-throughput genetic and chemical screens where a change in neutrophil number is an endpoint metric. Using the MCAM™ we have been able to, within minutes, acquire both enough data to create an automated algorithm and execute a biological experiment with statistical significance. Finally, we present this open-source software package which allows the user to train and evaluate a custom machine learning segmentation model and use it to localize zebrafish and analyze cell counts within the segmented region of interest. This software can be modified as needed for studies involving other zebrafish cell lineages using different transgenic reporter lines and can also be adapted for studies using other amenable model species.
Identifiants
pubmed: 38060605
doi: 10.1371/journal.pone.0295711
pii: PONE-D-23-28955
pmc: PMC10703246
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0295711Subventions
Organisme : NIEHS NIH HHS
ID : P42 ES031009
Pays : United States
Organisme : NIH HHS
ID : R44 OD024879
Pays : United States
Informations de copyright
Copyright: © 2023 Efromson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Déclaration de conflit d'intérêts
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AB, TJJD, CD, JE, MH, PR, and VS are employees of, have a financial interest in, and contribute intellectual property to Ramona Optics Inc., which is commercializing the multicamera array microscope. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Références
Fish Shellfish Immunol. 2015 Aug;45(2):286-92
pubmed: 25956719
Microsc Res Tech. 2022 Jan;85(1):169-180
pubmed: 34369634
Am J Med. 1990 Nov;89(5):663-72
pubmed: 2239986
Neurotoxicology. 2009 Jan;30(1):52-8
pubmed: 18952124
J Cell Sci. 2011 Sep 15;124(Pt 18):3053-9
pubmed: 21868367
Rev Bras Hematol Hemoter. 2013;35(1):18-22
pubmed: 23580879
J Immunotoxicol. 2023 Dec;20(1):2176953
pubmed: 36788734
Transl Vis Sci Technol. 2020 Feb 18;9(2):11
pubmed: 32704417
Molecules. 2014 Feb 21;19(2):2390-409
pubmed: 24566310
Sci Transl Med. 2014 Feb 26;6(225):225ra29
pubmed: 24574340
Shock. 2014 Jun;41(6):491-8
pubmed: 24837201
Comput Biol Med. 2021 Aug;135:104572
pubmed: 34182331
Biol Open. 2021 Sep 15;10(9):
pubmed: 34472582
Elife. 2022 Dec 14;11:
pubmed: 36515989
Blood. 2006 Dec 15;108(13):3976-8
pubmed: 16926288
Nat Immunol. 2011 Jun 26;12(8):761-9
pubmed: 21706006
Healthc Technol Lett. 2019 Jul 17;6(4):103-108
pubmed: 31531224
Fish Shellfish Immunol. 2023 Jan;132:108466
pubmed: 36462742
Cell Tissue Res. 2010 Feb;339(2):437-48
pubmed: 20012648
Blood. 2011 Jan 27;117(4):e49-56
pubmed: 21084707
Med Phys. 2020 Jun;47(5):e148-e167
pubmed: 32418337
Elife. 2022 Sep 30;11:
pubmed: 36178806
Support Care Cancer. 2016 Oct;24(10):4377-83
pubmed: 27278272
Environ Int. 2021 Nov;156:106599
pubmed: 33993002
Birth Defects Res C Embryo Today. 2011 Sep;93(3):281-7
pubmed: 21932436
BMC Dev Biol. 2007 May 04;7:42
pubmed: 17477879
Br J Haematol. 2005 Jun;129(6):713-22
pubmed: 15952996
IEEE/ACM Trans Comput Biol Bioinform. 2016 Mar-Apr;13(2):326-40
pubmed: 27045831
Behav Brain Res. 2020 Aug 5;391:112625
pubmed: 32428631
Wiley Interdiscip Rev Dev Biol. 2018 May;7(3):e312
pubmed: 29436122
Bioinform Biol Insights. 2021 Aug 7;15:11779322211037770
pubmed: 34413636
Nat Photonics. 2023 May;17(5):442-450
pubmed: 37808252
J Biophotonics. 2018 Mar;11(3):
pubmed: 28851134
Nat Immunol. 2014 Jan;15(1):45-53
pubmed: 24270515
Blood. 2008 Jan 1;111(1):132-41
pubmed: 17875807
Immunity. 2021 Jul 13;54(7):1377-1391
pubmed: 34260886
Infect Immun. 2009 Nov;77(11):5190-201
pubmed: 19687200
Opt Express. 2019 Mar 4;27(5):7787-7802
pubmed: 30876336
Anticancer Drugs. 2015 Nov;26(10):1054-60
pubmed: 26267078
Aquat Toxicol. 2023 Jul;260:106578
pubmed: 37244123
Blood. 2000 Oct 1;96(7):2317-22
pubmed: 11001877
SN Comput Sci. 2021;2(3):160
pubmed: 33778771
Int J Biochem Cell Biol. 2022 Jul;148:106234
pubmed: 35667555
Nat Commun. 2023 Aug 15;14(1):4942
pubmed: 37582932
Zebrafish. 2015 Jun;12(3):238-42
pubmed: 25790244
Proc R Soc Lond B Biol Sci. 1980 Feb 29;207(1167):187-217
pubmed: 6102765
Blood. 2011 Jun 30;117(26):7014-20
pubmed: 21596856
Sci Total Environ. 2022 Feb 25;809:152102
pubmed: 34863748