Facing a camera, Lina Carlini (biophysicist, postdoctoral fellow in Manley’s lab, EPFL) presents her recent work and gives emphasis on the physical problem behind it. Tatjana Kleele (biochemist, postdoc in Manley’s lab, EPFL) comments with a few lines to explain the biological approach used to solve the problem. They are from different disciplines but meet through chemical biology.

Perspective by Tatjana Kleele

The development of super-resolution microsc op yand in particular single molecule localization microscopy (SMLM) has opened entirely new possibilities to gain insight into cell biology at the scale of individual proteins. However, SMLM relies critically on probes that can photoswitch, i.e. transition between bright and dark states, so that their molecular positions can be determined with high precision. For reliable photoswitching, many small molecule probes depend on buffers that can compromise cell viability. here, we relied for photoswitching on the endogenous reducing and oxidizing agents present in various cellular compartments. These probes, which densely label organelles and yield many photons, are valuable for acquiring live-cell SMLM videos, while minimizing damage to the cell.This idea of exploiting the cell’s chemistry to induce single molecule photoswicthing was further expanded. We chemically transformed cell-permeable rhodamine dyes into their non-fluorescent leuco-rhodamine form. This transformation is reversible and occurs spontaneously through oxidation in a living cell. Labeling with this leuco-rhodamine dye increases single-molecule density, which is advantageous for live-cell SMLM since higher molecular densities yield improved spatial and temporal resolutions. In addition, the order of magnitude increase in single molecules is benefical for single particle tracking (spt) experiments. We have just described two examples of how intracellular reactions can promote molecular transitions between different optical states. In the future, the design of such photoswitches can advance live-cell compatible SMLM, and also serve as single molecule sensors.

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