Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

Fedor V. Subach, George H. Patterson, Suliana Manley, Jennifer M. Gillette, Jennifer Lippincott-Schwartz, Vladislav V. Verkhusha

Research output: Contribution to journalArticle

406 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)153-159
Number of pages7
JournalNature Methods
Volume6
Issue number2
DOIs
StatePublished - Feb 9 2009

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Subach, F. V., Patterson, G. H., Manley, S., Gillette, J. M., Lippincott-Schwartz, J., & Verkhusha, V. V. (2009). Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nature Methods, 6(2), 153-159. https://doi.org/10.1038/nmeth.1298