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High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding
Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveal...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683748/ https://www.ncbi.nlm.nih.gov/pubmed/31289126 http://dx.doi.org/10.1083/jcb.201812081 |
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author | Bykov, Yury S. Cohen, Nir Gabrielli, Natalia Manenschijn, Hetty Welsch, Sonja Chlanda, Petr Kukulski, Wanda Patil, Kiran R. Schuldiner, Maya Briggs, John A.G. |
author_facet | Bykov, Yury S. Cohen, Nir Gabrielli, Natalia Manenschijn, Hetty Welsch, Sonja Chlanda, Petr Kukulski, Wanda Patil, Kiran R. Schuldiner, Maya Briggs, John A.G. |
author_sort | Bykov, Yury S. |
collection | PubMed |
description | Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique. |
format | Online Article Text |
id | pubmed-6683748 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-66837482019-08-08 High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding Bykov, Yury S. Cohen, Nir Gabrielli, Natalia Manenschijn, Hetty Welsch, Sonja Chlanda, Petr Kukulski, Wanda Patil, Kiran R. Schuldiner, Maya Briggs, John A.G. J Cell Biol Research Articles Genetic screens using high-throughput fluorescent microscopes have generated large datasets, contributing many cell biological insights. Such approaches cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) reveals detailed cellular ultrastructure but requires time-consuming sample preparation, limiting throughput. Here we describe a robust method for screening by high-throughput EM. Our approach uses combinations of fluorophores as barcodes to uniquely mark each cell type in mixed populations and correlative light and EM (CLEM) to read the barcode of each cell before it is imaged by EM. Coupled with an easy-to-use software workflow for correlation, segmentation, and computer image analysis, our method, called “MultiCLEM,” allows us to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate several uses for MultiCLEM with 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher throughput, and can be used in multiple ways to enable EM to become a powerful screening technique. Rockefeller University Press 2019-08-05 2019-07-09 /pmc/articles/PMC6683748/ /pubmed/31289126 http://dx.doi.org/10.1083/jcb.201812081 Text en © 2019 MRC Laboratory of Molecular Biology https://creativecommons.org/licenses/by/4.0/This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Articles Bykov, Yury S. Cohen, Nir Gabrielli, Natalia Manenschijn, Hetty Welsch, Sonja Chlanda, Petr Kukulski, Wanda Patil, Kiran R. Schuldiner, Maya Briggs, John A.G. High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title | High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title_full | High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title_fullStr | High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title_full_unstemmed | High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title_short | High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
title_sort | high-throughput ultrastructure screening using electron microscopy and fluorescent barcoding |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683748/ https://www.ncbi.nlm.nih.gov/pubmed/31289126 http://dx.doi.org/10.1083/jcb.201812081 |
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