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 Verkhusha, Samuel T. Hess

Research output: Contribution to journalArticle

91 Citations (Scopus)

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

Fingerprint

Actins
Fluorescence
Transferrin Receptors
Optical Imaging
Hemagglutinins
Cytoskeleton
Fluorescent Dyes
Microscopy
Membrane Proteins
Proteins
Fibroblasts
Membranes
red fluorescent protein

ASJC Scopus subject areas

  • Biophysics

Cite this

Gunewardene, M. S., Subach, F. V., Gould, T. J., Penoncello, G. P., Gudheti, M. V., Verkhusha, 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

Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. / Gunewardene, Mudalige S.; Subach, Fedor V.; Gould, Travis J.; Penoncello, Gregory P.; Gudheti, Manasa V.; Verkhusha, Vladislav; Hess, Samuel T.

In: Biophysical Journal, Vol. 101, No. 6, 21.09.2011, p. 1522-1528.

Research output: Contribution to journalArticle

Gunewardene, Mudalige S. ; Subach, Fedor V. ; Gould, Travis J. ; Penoncello, Gregory P. ; Gudheti, Manasa V. ; Verkhusha, Vladislav ; Hess, Samuel T. / Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. In: Biophysical Journal. 2011 ; Vol. 101, No. 6. pp. 1522-1528.
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