Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells

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.

Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

“…For instance, the emission maxima of MitoTracker Red and ER-Tracker Red differ by 16 nm. They can be distinguished using a ratiometric method by splitting their emission into a shortand a long-wavelength channel; the probe identity can then be determined from the intensity ratio of these two channels (30,32,40).…”

“…It has been further demonstrated that many conventional dyes can be used for super-resolution imaging based on photoswitching/bleaching and localization of single molecules (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Using single-molecule-based super-resolution methods, 3D resolutions down to approximately 10 nm have been demonstrated for fixed samples (21)(22)(23)(24)(25)(26), and live-cell imaging has also been achieved with spatial resolutions of 20-60 nm at time resolutions ranging from 0.5 s to 1 min (27)(28)(29)(30)(31)(32).…”

Fluorescence nanoscopy. Methods and applications

“…In the first application of PALM to living cells, the Hess group showed that influenza hemagglutinin (HA), a protein associated with cholesterolrich membrane domains, resides in irregular clusters on length scales from few nm up to many micrometres; furthermore, these observations discarded the hypothesis that line tension (due to lipid fluid-fluid phase coexistence) is responsible for the shape of the domains [188]. PALM imaging of live fibroblasts has also demonstrated that both transferrin receptor (TfR) and HA are correlated with actin while TfR and HA are uncorrelated, providing with the first direct evidence that TfR and HA live in distinctly different domains and also suggesting a direct interaction of membrane proteins with the actin skeleton [128].…”

“…PS-CFP2 (converts from cyan to green [137]) and PSmOrange (converts from orange to red [138]) also show good spectroscopic properties and may be used to simultaneously tag two intracellular proteins. PAmKate is the farthest red-shifted photoactivatable protein [128], which facilitated multicolour PALM of cells simultaneously expressing Dendra2, PAmCherry1 and PAmKatetagged proteins with a single excitation wavelength [128].…”

Bright light, better labels

“…Researchers led by Sam Hess showed 12 that three fluorophores that all emit in the orange-red wavelengths could be distinguished from each other. Conventional microscopy would not be able to separate, say, a greenish-yellow label that emits two green photons for each red one from an orangish-yellow label that emits one green photon for each red one, but super-resolution microscopy can distinguish such signals because emitted photons are attributed to individual proteins.…”