Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Fluorescent Dyes / chemistry*
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods*
  • Mutagenesis, Site-Directed
  • Photochemical Processes

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • red fluorescent protein