Live-cell STED microscopy enables 50 nm resolution imaging with preserved cell proliferation.

Stimulated emission depletion (STED) microscopy is a super-resolution imaging technique that uses a high light dose to surpass the diffraction limit. The excellent spatiotemporal resolution achieved by STED, combined with its nontoxic labeling, facilitates super-resolution imaging in living cells. However, the use of high-intensity lasers, along with repeated fluorophore excitation-depletion cycles, may cause phototoxic effects. In this study, we examined the invasiveness of live-cell STED microscopy to validate its use. Investigating cell proliferation is among the best strategies for detecting and quantifying potential phototoxic effects. Therefore, we studied long-term (20 h) cell proliferation and survival after high-resolution (50 nm) STED imaging using a 775 nm depletion beam. We observed no significant differences in proliferation and mortality rates between STED- and non-STED-imaged control cells for various human cell lines (U2OS, HeLa, and RPE-1), with STED imaging performed on different cellular structures (nuclear pore complex, Golgi, actin, and mitochondria). Importantly, the STED-imaged cells showed no significant mitotic delay compared to the control when timing the onset of mitosis. In addition to long-term effects, we measured short-term stress response by observing cytosolic calcium levels after high-resolution STED imaging and during low-resolution STED scanning, and found no significant stress. These results show the applicability of STED microscopy for noninvasive super-resolution imaging in living cells.