Freeze, Zoom, Enhance : Increasing precision and resolution of cryoCLEM

Abstract

Microscopes are a very important tool to study and visualise living systems. Two popular microscope techniques Fluorescence Microscopy (FM) and Electron Microscopy (EM) can be combined on the same sample, which provides complementary information called correlative light and electron microscopy (CLEM). This thesis describes improvements in CLEM-techniques of cryofixed samples.The discovery of the bright fluorescence of uranyl acetate is reported, after cooling with liquid nitrogen. The fluorescent signal of uranyl-acetate makes it very straightforward to find back regions in the EM. Furthermore, accurately alignment of the FM and EM images could be achieved using this phenomenon.The optical resolution of cryoFM is limited by practical restrictions. This results in a large resolution-gap between cryoFM and cryoEM, which can make it difficult to precisely interpret cryoCLEM data. In this thesis, super-resolution on cryosamples is developed. To do so three major challenges had to be overcome: increased drift, sample damage due to high intensity lasers and the unknown behaviour of fluorescent proteins under cryo-conditions. Overcoming these challenges allowed the performance of super-resolution cryoCLEM (SR-cryoCLEM), with a localisation accuracy of 30 nm and structural resolution of 50-100 nm, an increase of 4-8 times. </p