Photoactivatable mCherry for high-resolution two-color fluorescence microscopy

By F. Subach, Georges H. Patterson, Suliana Manley, J. Gillette, Jennifer Lippincott-Schwartz, V. Verkhusha. Nature Methods, 2009

627 citations recorded by the source corpus.

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.

Subfields: Biophysics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/782bced75a520fb8aec32e3f3b457ded9cc50af6

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Citations in the corpus

Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging (2011, neutral): “29–30, Table 3).”
Fast three-dimensional super-resolution imaging of live cells (2011, background): “Therefore, although photoactivation-facilitated high-density particle tracking has proven powerful for probing molecular motions in living cell”
Super resolution fluorescence microscopy (2009, neutral): “This problem was overcome by a recently developed Dronpa variant, bsDronpa, with a blue-shifted emission (61), and the dark-to-red switching PAmCherry, which is nonfluorescent prior to photoactivation (62).”
Fluorescent proteins and their applications in imaging living cells and tissues. (2010, background): “To date, successful PALM imaging has been demonstrated for PA-GFP (165, 166), Dronpa (395), PS-CFP2 (395), mEos2 (281), Dendra2 (281), and PA-mCherry1 (417).”
A guide to super-resolution fluorescence microscopy (2010, methodology): “Two-color applications on biological samples have been demonstrated for most approaches (Bates et al., 2007; Bock et al., 2007; Shroff et al., 2007; Bossi et al., 2008; Gunkel et al., 2009; Subach et al., 2009; van de Linde et al., 2009) and has been successfully used to map vertebrate kinetochore c”