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

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.

Pubmed ID: 19169259

Authors

  • Subach FV
  • Patterson GH
  • Manley S
  • Gillette JM
  • Lippincott-Schwartz J
  • Verkhusha VV

Journal

Nature methods

Publication Data

February 30, 2009

Associated Grants

  • Agency: NIGMS NIH HHS, Id: GM070358
  • Agency: NIGMS NIH HHS, Id: GM073913
  • Agency: NIGMS NIH HHS, Id: R01 GM070358
  • Agency: NIGMS NIH HHS, Id: R01 GM070358-05
  • Agency: NIGMS NIH HHS, Id: R01 GM073913
  • Agency: NIGMS NIH HHS, Id: R01 GM073913-04

Mesh Terms

  • Fluorescent Dyes
  • Luminescent Proteins
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • Photochemical Processes