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 journalArticlepeer-review

450 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 - 2009

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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