DOI: 10.1101/515841Jan 9, 2019Paper

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

BioRxiv : the Preprint Server for Biology
Yury BykovJohn A.G. Briggs

Abstract

Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can...Continue Reading

Related Concepts

Cytology
Electron Microscopy
Microscopy, Fluorescence
Computer Software
Yeasts
Fluorescence Angiography
Fluorophore
Genetic Screening (Procedure)
High Throughput Analysis
Population Group

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