UNIT 4.32 Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule. Issue 1 (19th June 2017)

• Record Type: Journal Article
• Authors: Saurabh, Saumya; Perez, Adam M.; Comerci, Colin J.; Shapiro, Lucy; Moerner, W. E.
• Is Part Of: Current protocols in cell biology. Volume 75:Issue 1(2017)
• Page Start: 4.32.1
• Page End: 4.32.22
• Publication Date: 2017-06-19
• Subjects: fluorogenic -- fluoromodule -- bacteria -- photostable -- STED -- single‐protein tracking; Cytology -- Laboratory manuals; Cytological Techniques; Cytology; Periodicals; 571.6028
• DOI: 10.1002/cpcb.21
• Languages: English
• ISSNs: 1934-2500

Abstract

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. © 2017 by John Wiley & Sons, Inc.