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.
Subach
Fedor V
FV
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.
Patterson
George H
GH
Manley
Suliana
S
Gillette
Jennifer M
JM
Lippincott-Schwartz
Jennifer
J
Verkhusha
Vladislav V
VV
D005456:Fluorescent Dyes / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D055668:Photochemical Processes; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein