Repository logo
 

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

Published version
Peer-reviewed

Type

Article

Change log

Authors

Bykov, Yury S 
Cohen, Nir 
Gabrielli, Natalia 
Manenschijn, Hetty 
Welsch, Sonja 

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.

Description

Keywords

Cell Wall, Fluorescent Dyes, Green Fluorescent Proteins, High-Throughput Screening Assays, Microscopy, Electron, Microscopy, Fluorescence, Mitochondria, Osmotic Pressure, Peroxisomes, Phenotype, Saccharomyces cerevisiae

Journal Title

The Journal of Cell Biology

Conference Name

Journal ISSN

0021-9525
1540-8140

Volume Title

218

Publisher

Rockefeller University Press
Sponsorship
This work was financially supported by grants from the Deutsche Forschungsgemeinschaft (SFB1129 Z2 to J.A.G. Briggs), EMBL (to J.A.G. Briggs), the Medical Research Council (MC_UP_1201/16 to J.A.G. Briggs), and the German Ministry of Education and Research (031A605 to K.R. Patil). The Schuldiner laboratory is supported by the European Research Council CoG 646604 Peroxisystem, the Deutsche Forschungsgemeinschaft (grant SFB1190 and a Deutsch-Israelische Projektkooperation [DIP] collaborative grant). N. Gabrielli was supported by the EMBL interdisciplinary postdoctoral program. M. Schuldiner is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics.