Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins

Abstract

We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzylguanine-substituted organic dyes, for labeling. Tetramethylrhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells.

Figure 1

Schematic of the labeling approach used in this study: A fusion protein of the protein of interest and hAGT is expressed in the cell (left). On incubation, the substrate TMRStar diffuses through the membrane and binds covalently to hAGT, leading to a specific fluorescence labeling of the protein of interest.

Figure 2

Subdiffraction-resolution STED imaging of gap junctions in living PtK2-cells. (A and B) Confocal and STED-image, respectively, of Connexin-43 fused to hAGT and labeled with TMR (raw data). Scale bar = 1 μm. (C) Profiles along the marked line in A and B that show that individual clusters can be discerned by STED but are not separated by confocal microscopy. Considering the finite extent of the protein agglomerations, a histogram (D) of the FWHM of 74 measured line profiles through individual clusters indicates a resolution <40 nm. (E) Time lapse STED imaging of the movement of Connexin-43 within the membrane. Scale bar = 500 nm.

Figure 3

Comparison of labeling with the tagging protein hAGT and substrate TMR-Star versus labeling with the yellow fluorescent protein Citrine for live cell STED imaging. (A) Confocal versus (B) STED image of vimentin labeled with TMR via hAGT, showing that STED renders the vimentin network much more clearly than its confocal counterpart. (C and D) Analogous Citrine-fused structure. Caveolin labeled by TMR via hAGT is shown in E, whereas the data in F is recorded by labeling with Citrine. Again, in the STED part of the images, individual clusters can be resolved. All images display raw data that can be further processed by image deconvolution not applied here. Scale bars = 1 μm.

Figure 4

Continuous wave (CW) STED imaging of tubulin-associated protein MAP2 labeled with hAGT-TMR. The magnifications point out the superior resolution obtainable through CW STED that simplifies the setup compared to the pulsed STED approach. Single tubules can be distinguished in the STED image, whereas in the confocal counterpart, they seem to form bundles. Scale bar = 1 μm.