Abstract
Live cell fluorescence imaging is the method of choice for studying dynamic processes, such as nuclear transport, vesicular trafficking, and virus entry and egress. However, endogenous cellular autofluorescence masks a useful fluorescence signal, limiting the ability to reliably visualize low-abundance fluorescent proteins. Here, we employed synchronously amplified fluorescence image recovery (SAFIRe), which optically alters ground versus photophysical dark state populations within fluorescent proteins to modulate and selectively detect their background-free emission. Using a photoswitchable rsFastLime fluorescent protein combined with a simple illumination and image-processing scheme, we demonstrate the utility of this approach for suppressing undesirable, unmodulatable fluorescence background. Significantly, we adapted this technique to different commercial wide-field and spinning-disk confocal microscopes, obtaining >10-fold improvements in signal to background. SAFIRe allowed visualization of rsFastLime targeted to mitochondria by efficiently suppressing endogenous autofluorescence or overexpressed cytosolic unmodulatable EGFP. Suppression of the overlapping EGFP signal provided a means to perform multiplexed imaging of rsFastLime and spectrally overlapping fluorophores. Importantly, we used SAFIRe to reliably visualize and track single rsFastLime-labeled HIV-1 particles in living cells exhibiting high and uneven autofluorescence signals. Time-lapse SAFIRe imaging can be performed for an extended period of time to visualize HIV-1 entry into cells. SAFIRe should be broadly applicable for imaging live cell dynamics with commercial microscopes, even in strongly autofluorescent cells or cells expressing spectrally overlapping fluorescent proteins.
Highlights
Live cell fluorescence imaging is the method of choice for studying dynamic processes, such as nuclear transport, vesicular trafficking, and virus entry and egress
Using a photoswitchable rsFastLime fluorescent protein combined with a simple illumination and image-processing scheme, we demonstrate the utility of this approach for suppressing undesirable, unmodulatable fluorescence background
To implement synchronously amplified fluorescence image recovery (SAFIRe) imaging on commercial fluorescence microscopes, we utilized rsFastLime [18, 19], a PS-Fluorescent proteins (FPs) with an extinction coefficient and fluorescence quantum yield comparable to those of EGFP
Summary
PS-FPs can be efficiently photoisomerized and their signals selectively recovered because of their high absorption crosssections and large reverse isomerization quantum yields These properties allow considerable alteration of emissive and dark populations by mild illumination intensity (ϳ10 W/cm2) [21, 37]. Bright false-positive autofluorescent puncta were suppressed, enabling reliable identification of single viral particles containing IN-rsFastLime (Fig. 3D) Both autofluorescent and IN-EGFP–labeled pseudovirus signals were suppressed by SAFIRe (Fig. 3G). We observed about 30% loss of the original intensity after 100 cycles because of irreversible photobleaching of rsFastLime population This partial loss of rsFastLime signal within the viral particles was not associated with cellular toxicity, as evidenced by continued endocytic activity and unaltered cell morphology
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