Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells.
| Autor: | Gunewardene MS; Department of Physics and Astronomy and Institute for Molecular Biophysics, University of Maine, Orono, Maine, USA., Subach FV, Gould TJ, Penoncello GP, Gudheti MV, Verkhusha VV, Hess ST |
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| Jazyk: | angličtina |
| Zdroj: | Biophysical journal [Biophys J] 2011 Sep 21; Vol. 101 (6), pp. 1522-8. Date of Electronic Publication: 2011 Sep 20. |
| DOI: | 10.1016/j.bpj.2011.07.049 |
| Abstrakt: | 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. (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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