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Chemphyschem. 2014 Mar 17;15(4):687-95. doi: 10.1002/cphc.201300757. Epub 2013 Nov 5.

Localization microscopy using noncovalent fluorogen activation by genetically encoded fluorogen-activating proteins.

Author information

1
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh PA 15213 (USA); Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213 (USA); Current address: Sharp Edge Laboratories, Inc., Pittsburgh PA 15203 (USA).

Abstract

The noncovalent equilibrium activation of a fluorogenic malachite green dye and its cognate fluorogen-activating protein (FAP) can produce a sparse labeling distribution of densely tagged genetically encoded proteins, enabling single molecule detection and super-resolution imaging in fixed and living cells. These sparse labeling conditions are achieved by control of the dye concentration in the milieu, and do not require any photoswitching or photoactivation. The labeling is achieved by using physiological buffers and cellular media, in which additives and switching buffers are not required to obtain super-resolution images. We evaluate the super-resolution properties and images obtained from a selected FAP clone fused to actin, and show that the photon counts per object are between those typically reported for fluorescent proteins and switching-dye pairs, resulting in 10-30 nm localization precision per object. This labeling strategy complements existing approaches, and may simplify multicolor labeling of cellular structures.

KEYWORDS:

fluorescence; imaging agents; localization microscopy; proteins; super resolution

PMID:
24194371
PMCID:
PMC3967550
DOI:
10.1002/cphc.201300757
[Indexed for MEDLINE]
Free PMC Article
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