Article abstract
Nature Methods 6, 153 - 159 (2009)
Published online: 25 January 2009 | Corrected online: 26 February 2009 | doi:10.1038/nmeth.1298
There is an Erratum (April 2009) associated with this Article.
There is an Erratum (April 2009) associated with this Article.
Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
Fedor V Subach1,3, George H Patterson2,3, Suliana Manley2, Jennifer M Gillette2, Jennifer Lippincott-Schwartz2 & Vladislav V Verkhusha1
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 
200 nm clusters of transferrin receptor and clathrin light chain at 25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.
- 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.
- Section on Organelle Biology, Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
- These authors contributed equally to this work.
Correspondence to: Vladislav V Verkhusha1 e-mail: vverkhus@aecom.yu.edu
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