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Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

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Integrative Biology Vol -6

Integrative Biology Vol -5

Integrative Biology Vol -4

Integrative Biology Vol -3

Integrative Biology Vol -2

Integrative Biology Vol -1

Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

Content Provider	Royal Society of Chemistry (RSC)
Author	Chapagain, Prem P. Jimenez, Ralph Dean, Kevin M. Regmi, Chola K. Davis, Lloyd M. Gerstman, Bernard S. Palmer, Amy E. Lubbeck, Jennifer L.
Copyright Year	2015
Abstract	Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities.
Starting Page	263
Ending Page	273
Page Count	11
File Format	HTM / HTML PDF
ISSN	17579694
Volume Number	7
Issue Number	2
Journal	Integrative Biology
DOI	10.1039/c4ib00251b
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Kriek Molecular dynamics Fluorescence Photobleaching HeLa Library Mutant MCherry
Content Type	Text
Resource Type	Article
Subject	Medicine Biochemistry Biophysics

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Microfluidics-Based Selection of Red-Fluorescent Proteins with Decreased Rates of Photobleaching

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Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

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Microfluidics-Based Selection of Red-Fluorescent Proteins with Decreased Rates of Photobleaching

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