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.
Highlights
Functional studies can be extended from individual proteins to a proteome-wide level using high content screens relying on genetic tools, fluorescent light microscopy (LM), and automated workflows
4 H and S5 B), suggesting that overexpression of these two proteins rescues the Δdnm1 mitochondrial phenotype without side effects affecting cellular ultrastructure. As result of this screen in which we combined conventional LM screening and a secondary ultrastructure characterization using MultiCLEM, we identified Mmo1 and Ydr366c as potential factors that might play an additional role in establishing mitochondrial morphology
Correlative microscopy To perform in-resin correlative light and EM (CLEM), we focused on peroxisomes
Summary
Functional studies can be extended from individual proteins to a proteome-wide level using high content screens relying on genetic tools, fluorescent light microscopy (LM), and automated workflows. Budding yeast (Saccharomyces cerevisiae, hereafter referred to as yeast) is a widely used model organism for such high-throughput studies. Easy and scalable genetic manipulation has allowed the creation of tools such as systematic deletion libraries and GFP collections (Giaever et al, 2002; Huh et al, 2003; Yofe et al, 2016; Weill et al, 2018). Combined with automatic fluorescence microscopy, these systematic libraries help to address a large variety of questions in cell biology (Ohya et al, 2005; Cohen and Schuldiner, 2011; Breker et al, 2014). EM has suffered, until now, from very low throughput
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