Super Resolution Live Cell Imaging on Electron Microscopy Grids for Correlation with Cryogenic Electron Tomography

Sammanfattning

Correlated light and electron microscopy (CLEM) combines the molecular specificity of fluorescence microscopy (FM) with the single digit nanometer scale structural information of electron microscopy (EM). To reduce the large resolution gap, causing ambiguities in the correlation, between the modalities, CLEM workflows increasingly incorporate super resolution (SR) FM. Traditionally, SR-FM in CLEM is performed under cryogenic conditions to ensure sample stability during correlation. However, performing SR-FM under cryogenic conditions imposes limitations on illumination intensity and alters fluorophore photophysics. This thesis investigates whether the SR-FM part of the CLEM workflow can be performed at room temperature using U2OS cells expressing vimentin–rsEGFP2 on EM grids. RESOLFT and STED imaging were performed prior to vitrification. RESOLFT imaging on grids was feasible with standard optical intensities, although it yielded lower resolution than on glass coverslips and was affected by several grid related challenges. In contrast, STED led to fluorophore bleaching, likely due to reflections and heat accumulation in the metallic grid dueto the high intensity of the depletion laser, making STED incompatible with the EM grids under the tested conditions. After vitrification, cryogenic fluorescence microscopy (cryoFLM) and electron tomography (cryoET) confirmed that cells on EM grids previously imaged at room temperature could be localized, demonstrating that room-temperature RESOLFT is compatible with cryoET. However, further work is required to establish whether SR imaging at room temperature can be reliably correlated with cryoET.