Open AccessSuper‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule
Author(s) -
Saurabh Saumya,
Perez Adam M.,
Comerci Colin J.,
Shapiro Lucy,
Moerner W. E.
Publication year - 2017
Publication title -
current protocols in cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.149
H-Index - 38
eISSN - 1934-2616
pISSN - 1934-2500
DOI - 10.1002/cpcb.21
Subject(s) - caulobacter crescentus , fluorescence microscope , bacteria , microscopy , live cell imaging , fluorescent protein , bacterial cell structure , protein tag , fluorescence , cytosol , biology , green fluorescent protein , genetically engineered , microbiology and biotechnology , biophysics , biochemistry , chemistry , bacterial protein , cell , enzyme , gene , genetics , fusion protein , recombinant dna , physics , quantum mechanics , optics
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. © 2017 by John Wiley & Sons, Inc.