Effects of base temperature, immersion medium, and EM grid material on devitrification thresholds in cryogenic...

Cryogenic correlative light and electron microscopy (cryo-CLEM) is an imaging strategy that integrates specific molecular labeling and molecular resolution structural information. However, there is a resolution gap of more than two orders of magnitude between diffraction-limited fluorescence microscopy and electron microscopy (EM). Single-molecule localization microscopy (SMLM) performed at cryogenic temperatures promises to bridge this resolution gap. Nevertheless, the high excitation laser powers required for SMLM risk the devitrification of frozen biological samples, leading to perturbation of their native-like state. Here, we investigate how base cooling temperature, immersion medium, and EM grid support materials influence sample devitrification. Using finite element simulations and experimental validation, we show that a cryo-immersion medium enhances heat dissipation for carbon supports, while metallic supports in a cold nitrogen gas medium tolerate higher laser intensities due to lower base temperatures. Gold supports illuminated at 640nm exhibit markedly high laser thresholds, similar to silver-coated grids. Additionally, metallic supports maintain efficient heat dissipation in vacuum-based cryostats. Our findings provide quantitative insights that aid in optimization of cryo-SMLM setups for improved cryo-CLEM imaging.