期刊
NATURE METHODS
卷 6, 期 2, 页码 153-159出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1298
关键词
-
资金
- US National Institutes of Health [GM070358, GM073913]
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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