Korrelative Mikroskopie bei Flüssigstickstoff-Temperatur.
Creators: name: Boehm, Ulrike
Publisher: name: figshare
Year: 2021
Subjects: subject: Applied Physics
subject: 20599 Optical Physics not elsewhere classified
subject: FOS: Physical sciences

description: Cryo-electron tomography (cryo-ET) permits observing cellular structures in their native state with almost molecular resolution. However, to maintain the cellular integrity, samples have to be embedded in vitrified ice and kept at all times at cryo-temperatures, respectively the temperature of liquid nitrogen. Cryo-ET is ideal for structural analysis at the level of single organelles and, moreover, suitable to study the spatial arrangement of macromolecular aggregates of entire organisms. However, the search and identification of biological structures are limited because of their sensitivity to electron radiation. Furthermore, biological material exhibits a very low contrast in transmission electron micrographs. Correlative microscopy at cryo-conditions offers a possibility to locate regions of interest prior to exhaustive cryo-EM investigations. It combines the advantages of the fluorescence/light microscope with those of the electron microscope. Obtaining information about location and distribution of biological objects with the help of fluorescence/light microscopy provides a faster and less invasive route. The final aim of correlative microscopy is to locate structures with light microscopic techniques andto zoom in on them with the electron microscope. However, special cryo-holders have to be developed to observe cryo-samples in light microscopes at liquid nitrogen temperature. Aim of this work was the optimization of the workflow for correlative microscopy, to quantify important system parameters, and to further improve this method for a potential routine use. For the first time a confocal microscope was used to achieve a better resolution and thus a better co-localisation of light and electron microscopic images. The co-localisation was estimated to 0.7 μm, thus an improvement of 10 nm compared to epifluorescence microscopy. During the course of the experiments a new cryo-holder model (CryoStage2) was further improved and optimized. Moreover, the development of programs for the correlation of EM and LM images was included. A disadvantage of the present holder was identified and characterized. The liquid nitrogen supply system is responsible for drifts of the holder during investigations on the range of micrometers. These drifts complicate quantitative measurements, for example in photobleaching experiments. Bleaching experiments were performed,and a comparison was made for bleaching at room temperature and at liquid nitrogen temperature. It was found that the bleaching rate occurred four times faster at room temperature than at liquid nitrogen temperature. According to this, fluorophores are more photostable at liquid nitrogen temperature, which could be beneficial for various applications where long exposure times are required. Finally, it is possible to combine the correlative microscopy approach with other techniques like focussed ion beam thinning of frozen hydrated samples and x-ray tomography to identify region of interest and to target specific structures in a fast and non-invasive way.