Two-color live-cell STED nanoscopy by click labeling with cell-permeable fluorophores

Abstract

STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are required. In addition, the fluorophores should preferentially emit in the red spectral range to reduce potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. Here we demonstrate two-color STED imaging of living cells using various pairs of dyes that fulfill all of the above requirements. To this end, we combine click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, enabling long-term STED imaging of different target structures in living specimens.

Bioorthogonal Caging-Group-Free Photoactivatable Probes for Minimal-Linkage-Error Nanoscopy

“…(c) PALM image of cells described in panel a, labeled with compound 4 . (d) COS-7 cells transiently expressing a vimentin-mCerulean3 fusion construct carrying a N116TAG mutation (for incorporating the UAA endo BCN- L -lysine) in vimentin. , The plasmids were provided by the Lemke laboratory (EMBL, Heidelberg). See Figure S14 for detailed maps of tRNAPyl/NESPylRSAF, and vimentin N116TAG -mCerulean3 plasmids.…”

“…Taken together, these results highlight the diverse applicability of PaX-Tz dyads: while the probes featuring alkene radical traps (e.g., 3) require much less UV Next, COS-7 cells were prepared (Figure 5d), transiently expressing a vimentin-mCerulean3 fusion construct 49 carrying a N116TAG mutation (for incorporating the UAA endo BCN-L-lysine, Figure S14) in the 1A coil fragment of the vimentin head domain. 50,51 Initial staining with 3 (Figure 5d) demonstrated bright labeling of the mCerulean3-tagged vimentin filaments after photoactivation, with a minimal background that can be attributed to nonincorporated UAA, free or bound to tRNA, or unreacted PaX-Tz in lipophilic compartments. Using this strategy, cells were further imaged by PALM microscopy (Figure 5e and Figure S15) for a subset of our dyads.…”

Pushing the Resolution Limit of Stimulated Emission Depletion Optical Nanoscopy

“…It is important to note that the resolution achieved in any fluorescence microscopy is contingent upon the location accuracy of the fluorescent probe used to label the molecule of interest. Therefore, we also discuss recent efforts to develop labeling strategies that offer enhanced position accuracy down to the nanometer scale [38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55]. The development of STED nanoscopy has seen significant strides made, but there are still opportunities for further advancement.…”

“…Thirdly, we present recent endeavors to improve temporal resolution by parallelizing STED donut beams [ 36 , 37 ] or by conditionally triggering STED observations [ 19 ]. Finally, we delve into recent developments in labeling strategies, which bear a close relationship to spatial resolution [ 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 ]. By examining the latest progress in each of these domains, we aim to provide an up-to-date and comprehensive overview of the present state of STED nanoscopy, as well as its potential in future research.…”

“…Sci. 2024, 25, x FOR PEER REVIEW 15 of 20 discuss recent efforts to develop labeling strategies that offer enhanced position accuracy down to the nanometer scale[38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55].…”