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Nat Methods. 2009 Feb;6(2):153-9. doi: 10.1038/nmeth.1298. Epub 2009 Jan 25.

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.

Author information

1
Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, USA.

Erratum in

  • Nat Methods. 2009 Apr;6(4):311.

Abstract

The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP-tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified < or =200 nm clusters of transferrin receptor and clathrin light chain at < or =25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

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PMID:
19169259
PMCID:
PMC2901231
DOI:
10.1038/nmeth.1298
[Indexed for MEDLINE]
Free PMC Article

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