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J Cell Biol. 2019 Aug 5;218(8):2797-2811. doi: 10.1083/jcb.201812081. Epub 2019 Jul 9.

High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Author information

1
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
2
Structural Studies Division, Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK.
3
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
4
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
5
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel maya.schuldiner@weizmann.ac.il.
6
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany john.briggs@mrc-lmb.cam.ac.uk.
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Contributed equally

Abstract

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.

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