Primary Light-Induced Reaction Steps of Reversibly Photoswitchable Fluorescent Protein Padron0.9 Investigated by Femtosecond Spectroscopy

The reversible photoswitching of the photochromic fluorescent protein Padron0.9 involves a <i>cis–trans</i> isomerization of the chromophore. Both isomers are subjected to a protonation equilibrium between a neutral and a deprotonated form. The observed pH dependent absorption spectra require at least two protonating groups in the chromophore environment modulating its proton affinity. Using femtosecond transient absorption spectroscopy, we elucidate the primary reaction steps of selectively excited chromophore species. Employing kinetic and spectral modeling of the time dependent transients, we identify intermediate states and their spectra. Excitation of the deprotonated <i>trans</i> species is followed by excited state relaxation and internal conversion to a hot ground state on a time scale of 1.1–6.5 ps. As the switching yield is very low (Φ_{<i>trans→cis</i>} = 0.0003 ± 0.0001), direct formation of the <i>cis</i> isomer in the time-resolved experiment is not observed. The reverse switching route involves excitation of the neutral <i>cis</i> chromophore. A strong H/D isotope effect reveals the initial reaction step to be an excited state proton transfer with a rate constant of <i>k</i>_H = (1.7 ps)⁻¹ (<i>k</i>_D = (8.6 ps)⁻¹) competing with internal conversion (<i>k</i>_ic = (4.5 ps)⁻¹). The deprotonated excited <i>cis</i> intermediate relaxes to the well-known long-lived fluorescent species (<i>k</i>_r = (24 ps)⁻¹). The switching quantum yield is determined to be low as well, Φ_{<i>cis→trans</i>} = 0.02 ± 0.01. Excitation of both the neutral and deprotonated <i>cis</i> chromophores is followed by a ground state proton transfer reaction partially re-establishing the disturbed ground state equilibrium within 1.6 ps (deuterated species: 5.6 ps). The incomplete equilibration reveals an inhomogeneous population of deprotonated <i>cis</i> species which equilibrate on different time scales

RESOLFT Nanoscopy of Fixed Cells Using a Z-Domain Based Fusion Protein for Labelling

<div><p>RESOLFT super-resolution microscopy allows subdiffraction resolution imaging of living cells using low intensities of light. It relies on the light-driven switching of reversible switchable fluorescent proteins (RSFPs). So far, RESOLFT imaging was restricted to living cells, because chemical fixation typically affects the switching characteristics of RSFPs. In this study we created a fusion construct (FLASR) consisting of the RSFP rsEGFP2 and the divalent form of the antibody binding Z domain from protein A. FLASR can be used analogous to secondary antibodies in conventional immunochemistry, facilitating simple and robust sample preparation. We demonstrate RESOLFT super-resolution microscopy on chemically fixed mammalian cells. The approach may be extended to other super-resolution approaches requiring fluorescent proteins in an aqueous environment.</p></div

RESOLFT super-resolution image of an entire CV-1 cell.

<p>The cell was decorated with primary antibodies against α-tubulin and FLASR. Scale bar: 5 μm.</p

Immunolabelling with FLASR.

<p>(A) Schematic of FLASR (ZZ-rsEGFP2_tandem) bound to an immunoglobulin protein. (B-D) Maximum intensity projections of confocal z-stacks of methanol fixed mammalian CV-1 cells immunolabelled with antibodies against β-actin (B), vimentin (C) and α-tubulin (D). Subsequently, purified FLASR (red) was used to decorate the primary antibodies. Nuclei were stained with DAPI (blue). Scale bars: 50 μm.</p

Immunolabelling with an M-rsEGFP2_tandem fusion protein.

<p>Maximum intensity projections of confocal microscopy z-stacks of methanol fixed CV-1 cells immunolabelled with antibodies against β-actin (A) and vimentin (B). The purified recombinant fusion protein M-rsEGFP2_tandem was used to decorate the primary antibodies (red). Nuclei were labelled with DAPI (blue). Scale bars: 50 μm.</p

RESOLFT nanoscopy of methanol fixed cells.

<p>Comparison of RESOLFT super-resolution microscopy and the corresponding confocal microscopy images of CV-1 cells decorated with primary antibodies against vimentin (A), α-tubulin (B) and the nuclear pore complex protein Nup153 (C). (D) Line-profiles of the fluorescence intensities recorded between the arrowheads in (A-C), as indicated (confocal: light blue; RESOLFT: red). The line profiles in (1–3) are averaged across five adjacent line profiles that were perpendicular across the respective filament. The distance between two adjacent line profiles was the edge length of one pixel. Scale bars: 1 μm.</p

Labelling of SNAP-, CLIP- and Halo-tagged proteins in chemically fixed and living budding yeast cells.

<p>(A) Chemically fixed cells expressing the respective self-labelling proteins targeted to the mitochondrial matrix (mtSNAP, mtCLIP, or mtHalo) were labelled. (B) Labelling of live yeast cells expressing the mitochondrial targeted self-labelling proteins using an electroporation protocol. (C) Live yeast cells expressing the indicated fusion proteins labelled by electroporation. Cells were labelled using commercially available TMR substrates. Yeast strains expressing Abp1-SNAP and Pil1-CLIP were created by epitope-tagging, while the other fusion constructs were plasmid encoded. Shown are maximum projections of confocal sections. Scale bar: 2 µm. </p

Live cell super-resolution microscopy and multi-colour microscopy using self-labelling proteins.

<p>(A) Living yeast cells expressing Pil1-CLIP at a near native level from the endogenous chromosomal locus were labelled by electroporation with Atto565-CLIP and imaged using confocal (left) and STED (right) microscopy. Inset: Intensity profile over the region marked with the arrow heads. (B) Dual colour labelling with the CLIP- and the Halo-tag. mtHalo was labelled with 6′-CR110-Halo and Pil1-CLIP was labelled with CLIP-Cell TMR-Star and imaged by epifluorescence microscopy. Scale bars: 2 µm. </p

STED super-resolution microscopy of mitochondria in the rectal <i>Muscularis externa</i> demonstrates high structural preservation of the stored paraffin-embedded tissue.

<p>STED recordings were performed on 2 µm thick dewaxed sections cut along the longitudinal axis of the rectum. (A) Left: STED overview image of a region of the inner circular layer of the rectal <i>Muscularis externa</i> decorated with an antiserum against Tom20. Right: Magnifications of the areas in the indicated dashed squares showing the distribution of TOM clusters within the mitochondria. (B–E) STED images of tissue sections decorated with antisera against Tom20 (B), Mic60 (mitofilin) (C), aconitase (D), and cyclophilin D (E). In each panel the confocal (top, left) and the corresponding STED image (top, right) is displayed. Bottom: Magnification of the STED image as indicated by a dashed square. Note the different distributions of the four proteins within the mitochondria. Scale bars: 20 µm (A, left); 1 µm (A, right) and (B–E, top); 200 nm (B–E, bottom).</p

STED super-resolution microscopy of archived human tissue samples stored for up to 17 years in a clinical repository.

<p>Representative images of tumor tissues stored at room temperature for less than 1 year (A), 11 years (B) or 17 years (C), were sectioned, dewaxed, decorated with an antiserum against Tom20 and imaged. Left: Representative confocal images. The same color table was used for the three images in order to visualize the relative staining efficiencies. Middle/Right: Comparison of STED (middle) and confocal (right) microscopy of tissue sections of different age. Here, the color tables were adjusted to the signal intensities obtained. Scale bars: 10 µm (left) and 1 µm (middle, right).</p