Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry

Beaton-Green, Lindsay A.; Rodrigues, Matthew A.; Lachapelle, Sylvie; Wilkins, Ruth

doi:10.1016/j.ymeth.2016.08.006

Cited by 7 publications

(4 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The EDF option on the ISX allows light from different focal planes to be imaged on the detector plane simultaneously. The EDF is a useful option that allows structures and probes, such as individual chromosomes in suspension (54,55) or cH2AX foci (56,57) to be focused onto a single two-dimensional image. This improves the ability to count spots at different focus depths within the cell.…”

Section: Discussionmentioning

confidence: 99%

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Rodrigues¹

2018

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

The in vitro micronucleus (MN) assay is a well‐established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide‐scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide‐scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re‐evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono‐ and bi‐nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well‐known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

show abstract

Section: Discussionmentioning

confidence: 99%

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Rodrigues¹

2018

Cytometry Pt A

Self Cite

View full text Add to dashboard Cite

show abstract

“…The ImageStream ®X Mark II (Amnis ® Flow Cytometry, Luminex, Seattle, WA, USA) combines the speed and statistical robustness of conventional flow cytometry with high-resolution imagery capabilities of microscopy in a single system 36 . IFC technology has been used to quantify autophagy 37 and extracellular vesicles 38 , to demonstrate diagnostic potential in acute leukemia 39 , for assessment of phagocytosis and NETosis 40 and to quantify DNA damage via the chromosomal aberration assay 41 . The ImageStream ®X Mark II is accompanied by a powerful image analysis software package (IDEAS ® ) which permits robust analysis strategies to be created.…”

Section: Introductionmentioning

confidence: 99%

The in vitro micronucleus assay using imaging flow cytometry and deep learning

et al. 2021

Self Cite

View full text Add to dashboard Cite

The in vitro micronucleus (MN) assay is a well-established assay for quantification of DNA damage, and is required by regulatory bodies worldwide to screen chemicals for genetic toxicity. The MN assay is performed in two variations: scoring MN in cytokinesis-blocked binucleated cells or directly in unblocked mononucleated cells. Several methods have been developed to score the MN assay, including manual and automated microscopy, and conventional flow cytometry, each with advantages and limitations. Previously, we applied imaging flow cytometry (IFC) using the ImageStream® to develop a rapid and automated MN assay based on high throughput image capture and feature-based image analysis in the IDEAS® software. However, the analysis strategy required rigorous optimization across chemicals and cell lines. To overcome the complexity and rigidity of feature-based image analysis, in this study we used the Amnis® AI software to develop a deep-learning method based on convolutional neural networks to score IFC data in both the cytokinesis-blocked and unblocked versions of the MN assay. We show that the use of the Amnis AI software to score imagery acquired using the ImageStream® compares well to manual microscopy and outperforms IDEAS® feature-based analysis, facilitating full automation of the MN assay.

show abstract

“…Some success has been seen with the DCA. However, there is still room for improvement in the processing of the samples to generate a solution of chromosomes that maintain their morphology through manipulation and do not become aggregated ( 55 , 56 ) .…”

Section: Imaging Flow Cytometrymentioning

confidence: 99%

Development of high-throughput systems for biodosimetry

Wilkins,

Beaton-Green

2023

Radiation Protection Dosimetry

View full text Add to dashboard Cite

Biomarkers for ionising radiation exposure have great utility in scenarios where there has been a potential exposure and physical dosimetry is missing or in dispute, such as for occupational and accidental exposures. Biomarkers that respond as a function of dose are particularly useful as biodosemeters to determine the dose of radiation to which an individual has been exposed. These dose measurements can also be used in medical scenarios to track doses from medical exposures and even have the potential to identify an individual’s response to radiation exposure that could help tailor treatments. The measurement of biomarkers of exposure in medicine and for accidents, where a larger number of samples would be required, is limited by the throughput of analysis (i.e. the number of samples that could be processed and analysed), particularly for microscope-based methods, which tend to be labour-intensive. Rapid analysis in an emergency scenario, such as a large-scale accident, would provide dose estimates to medical practitioners, allowing timely administration of the appropriate medical countermeasures to help mitigate the effects of radiation exposure. In order to improve sample throughput for biomarker analysis, much effort has been devoted to automating the process from sample preparation through automated image analysis. This paper will focus mainly on biological endpoints traditionally analysed by microscopy, specifically dicentric chromosomes, micronuclei and gamma-H2AX. These endpoints provide examples where sample throughput has been improved through automated image acquisition, analysis of images acquired by microscopy, as well as methods that have been developed for analysis using imaging flow cytometry.

show abstract

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry

Cited by 7 publications

References 22 publications

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

The in vitro micronucleus assay using imaging flow cytometry and deep learning

Development of high-throughput systems for biodosimetry

Contact Info

Product

Resources

About

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry

Cited by 7 publications

References 22 publications

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer

The in vitro micronucleus assay using imaging flow cytometry and deep learning

Development of high-throughput systems for biodosimetry

Contact Info

Product

Resources

About

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer

Automation of the in vitro micronucleus assay using the Imagestream^® imaging flow cytometer