Building a cryostate for Cryo fluorescence microscopy

Abstract

Fluorescence microscopy has been one of the most important tools for understanding how biological systems are organized and how they function. Over the last two decades, the resolution of fluorescence microscopy has been enhanced by the development of super resolution methods that extend the limit of optical microscopy beyond the diffraction limit, providing unprecedented levels of information on the organization of molecular networks in cells. These methods include Structured Illumination Microscopy (SIM), Stimulated Emission Depletion Microscopy (STED),and techniques based on the localization of individual fluorescent molecules: Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM), referred to collectively as Single Molecule Localization Microscopy (SMLM) techniques. Super-resolution methods usually involve the use of chemical fixation, which has been shown to introduce artefacts that can be visible at the resolutions achieved. An alternative to chemical fixation is cryofixation, which is a superior structural preservation method, but requires to preserve the specimen under cryogenic temperatures during the measurement, which requires a big adaption of the experimental set-up. Favorably, cryogenic temperatures slow down the photobleaching of fluorophores, which has the potential to improve the resolution. In this work, after a short introduction about fluorescence super resolution microscopy, the main topic will be how we built a setup for super resolution microscopy at cryogenic temperatures. After that, preliminary measurements with fluorescent dyes AF-647 (Alexa Fluor) and Atto-647N will be shown and discussed how their photophysical properties are affected by low temperatures.