TY - JOUR
T1 - Photoactivatable mCherry for high-resolution two-color fluorescence microscopy
AU - Subach, Fedor V.
AU - Patterson, George H.
AU - Manley, Suliana
AU - Gillette, Jennifer M.
AU - Lippincott-Schwartz, Jennifer
AU - Verkhusha, Vladislav V.
N1 - Funding Information:
We thank R. Tsien (University of California at San Diego) for providing the pRSETB-mCherry plasmid, J. Wiedenmann (University of Ulm) for providing plasmids encoding EosFP variants, J. Zhang for assistance with flow cytometry, O. Subach for assistance with cell culture and imaging, and E. Betzig and H. Hess for assistance with PALM experiments, analysis and discussion. This work was supported by grants GM070358 and GM073913 from the US National Institutes of Health to V.V.V.
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
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U2 - 10.1038/nmeth.1298
DO - 10.1038/nmeth.1298
M3 - Article
C2 - 19169259
AN - SCOPUS:59349110743
SN - 1548-7091
VL - 6
SP - 153
EP - 159
JO - Nature Methods
JF - Nature Methods
IS - 2
ER -