Novel reversibly switchable fluorescent proteins for RESOLFT and STED nanoscopy engineered from the bacterial photoreceptor YtvA.

The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limited switching speed. Therefore, RSFPs from other origins with improved properties need to be explored. Here, we report the development of two RSFPs based on the LOV domain of the photoreceptor protein YtvA from Bacillus subtilis. LOV domains obtain their fluorescence by association with the abundant cellular cofactor flavin mononucleotide (FMN). Under illumination with blue and ultraviolet light, they undergo a photocycle, making these proteins inherently photoswitchable. Our first improved variant, rsLOV1, can be used for RESOLFT imaging, whereas rsLOV2 proved useful for STED nanoscopy of living cells with a resolution of down to 50 nm. In addition to their smaller size compared to GFP-related proteins (17 kDa instead of 27 kDa) and their usability at low pH, rsLOV1 and rsLOV2 exhibit faster switching kinetics, switching on and off 3 times faster than rsEGFP2, the fastest-switching RSFP reported to date. Therefore, LOV-domain-based RSFPs have potential for applications where the switching speed of GFP-based proteins is limiting.

Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED.

Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes.

Multicolour Multilevel STED nanoscopy of Actin/Spectrin Organization at Synapses.

Superresolution fluorescence microscopy of multiple fluorophores still requires development. Here we present simultaneous three-colour stimulated emission depletion (STED) nanoscopy relying on a single STED beam at 620 nm. Toggling the STED beam between two or more power levels ("multilevelSTED") optimizes resolution and contrast in all colour channels, which are intrinsically co-aligned and well separated. Three-colour recording is demonstrated by imaging the nanoscale cytoskeletal organization in cultured hippocampal neurons. The down to ~35 nm resolution identified periodic actin/betaII spectrin lattices along dendrites and spines; however, at presynaptic and postsynaptic sites, these patterns were found to be absent. Both our multicolour scheme and the 620 nm STED line should be attractive for routine STED microscopy applications.

Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 µm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.