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

Biophys J. 2011 Sep 21;101(6):1522-8. doi: 10.1016/j.bpj.2011.07.049. Epub 2011 Sep 20.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actins / chemistry
  • Animals
  • Cell Survival
  • Color
  • Hemagglutinins / chemistry
  • Luminescent Proteins / chemistry*
  • Mice
  • Microscopy, Fluorescence / methods*
  • NIH 3T3 Cells
  • Receptors, Transferrin / chemistry

Substances

  • Actins
  • Hemagglutinins
  • Luminescent Proteins
  • Receptors, Transferrin