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Curr Protoc Cell Biol. 2017 Jun 19;75:4.32.1-4.32.22. doi: 10.1002/cpcb.21.

Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule.

Author information

1
Department of Chemistry, Stanford University, Stanford, California.
2
Department of Developmental Biology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California.
3
Department of Biology, Stanford University, Stanford, California.
4
Biophysics Program, Stanford University, Stanford, California.

Abstract

Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion.

KEYWORDS:

STED; bacteria; fluorogenic; fluoromodule; photostable; single-protein tracking

PMID:
28627757
PMCID:
PMC5768428
DOI:
10.1002/cpcb.21
[Indexed for MEDLINE]
Free PMC Article

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