Determination of factors that allow cryogenic nanoscopy with high power illumination without devitrification

Abstract

Cryogenic super-resolution fluorescence light microscopy, or nanoscopy, has been demonstrated to be useful to close the resolution gap in cryogenic correlative light and electron microscopy (CLEM). Importantly, under cryogenic conditions fundamental resolution barriers that are imposed by molecular motion and photobleaching on fluorescence microscopy are circumvented. The resolution gain is even higher in nanoscopic methods, due to higher obtainable resolution and slower acquisition speed, rendering cryo-nanoscopy promising even beyond CLEM. However, cryogenic nanoscopy is often limited by heating and devitrification of the sample through strong laser irradiation, with drastic differences in reported tolerable power densities. We therefore investigated the laser-induced heating in different setups for cryogenic nanoscopy by time-dependent finite-element simulations complemented with absorption measurements of mammalian cells. Laser-induced heating happened in milliseconds, precluding efficient sample preservation by most intermittent illuminations. Under moderate light densities used for single molecule localization microscopy, absorbance by mammalian cells was too weak to explain devitrification and heating is governed by absorption of supporting material. However, the much higher power densities used in stimulated emission depletion nanoscopy resulted in temperatures clearly above the devitrification temperature from absorption by cells alone, unless the sample was mounted on an efficient heat exchanger, such as a diamond.