Super-resolution fluorescence microscopy in cryogenic conditions

Abstract

Developing of super resolution light microscopy and fluorescent probes have opened the door to the undiscovered word of the living cells through labelling specific proteins of interest. Correlative light and electron microscopy (CLEM) is an imaging technique that combines light and electron microscopy to identify and track target labeled proteins while localizing them and gain more insight into the ultracellular structure at high resolution. Recent advanced sample preparation has made possible to vitrified samples without perturbing the nature structures as well as multiscale imaging of them in both modalities as chemical fixation of specimens for electron microscopy are not required. This work presents the sample preparation protocols at room and cryogenic temperature focusing on establishment of cryo fluorescence microscopy workflow using Chinese hamster ovary (CHO) cells expressing mGFP-GPI. Within this project we were able to rapidly freeze gird samples without any ice crystalline formation and transferred them safely at cryogenic temperature for imaging. Following, cryo Fluorescence images were acquired at various intensity excitations, illumination times and delay times to determine single molecule localization within nanometer scale while the thermal stability of the cryostat preserve steady during entire experiment.