Light-induced cell damage in live-cell super-resolution microscopy

TLDR

Super‑resolution microscopy can reveal hidden cellular details but requires high irradiation intensities that likely cause cellular damage. The study tests how irradiation parameters affect cell survival after single‑molecule localization microscopy and proposes strategies to reduce phototoxicity. The authors used single‑molecule localization microscopy conditions, varying irradiation intensity, mode, wavelength, dose, temperature, and labeling, and measured microtubule growth speed after irradiation. Lower wavelengths dramatically increase phototoxicity, causing fixation, membrane permeabilization, and cytoskeleton destruction; cells tolerate ~1 kW cm⁻² at 640 nm for minutes, but only ~50 J cm⁻² at 405 nm, underscoring the advantage of red fluorophores.

Abstract

Abstract Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm −2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm −2 , emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.