Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Mudalige S. Gunewardene, Fedor V. Subach, Travis J. Gould, Gregory P. Penoncello, Manasa V. Gudheti, Vladislav V. Verkhusha, Samuel T. Hess

Research output: Contribution to journalArticle

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Abstract

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 ∼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-β-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.

Original languageEnglish (US)
Pages (from-to)1522-1528
Number of pages7
JournalBiophysical journal
Volume101
Issue number6
DOIs
StatePublished - Sep 21 2011

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ASJC Scopus subject areas

  • Biophysics

Cite this

Gunewardene, M. S., Subach, F. V., Gould, T. J., Penoncello, G. P., Gudheti, M. V., Verkhusha, V. V., & Hess, S. T. (2011). Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. Biophysical journal, 101(6), 1522-1528. https://doi.org/10.1016/j.bpj.2011.07.049