Journal
BIOPHYSICAL JOURNAL
Volume 101, Issue 6, Pages 1522-1528Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2011.07.049
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Funding
- National Institute of Allergy and Infectious Diseases [A165459]
- National Institute of General Medical Sciences [GM073913, GM094713]
- National Science Foundation [CHE0722759]
- Maine Technology Institute [MTAF 1106, MTAF 2061]
- Maine Economic Improvement Fund
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Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only similar to 100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-beta-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.
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