Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research

rsEGFP2 enables fast RESOLFT nanoscopy of living cells

The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25–250 times faster recordings

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Despite remarkable developments in diffraction unlimited super-resolution microscopy, in vivo nanoscopy of tissues and model organisms is still not satisfactorily established and rarely realized. RESOLFT nanoscopy is particularly suited for live cell imaging because it requires relatively low light levels to overcome the diffraction barrier. Previously, we introduced the reversibly switchable fluorescent protein rsEGFP2, which facilitated fast RESOLFT nanoscopy (Grotjohann et al., 2012). In that study, as in most other nanoscopy studies, only cultivated single cells were analyzed. Here, we report on the use of rsEGFP2 for live-cell RESOLFT nanoscopy of sub-cellular structures of intact Drosophila melanogaster larvae and of resected tissues. We generated flies expressing fusion proteins of alpha-tubulin and rsEGFP2 highlighting the microtubule cytoskeleton in all cells. By focusing through the intact larval cuticle, we achieved lateral resolution of <60 nm. RESOLFT nanoscopy enabled time-lapse recordings comprising 40 images and facilitated recordings 40 µm deep within fly tissues

Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer ReviewedPostprint (author's final draft