Selecting optimal support grids for super-resolution cryogenic correlated light and electron microscopy

Abstract

Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude. Finally, we demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated cryo-microscopy.

Building a super-resolution fluorescence cryomicroscope

“…To create a single-molecule localization map that is suited for localizing structures of interest for subsequent cryoEM inspection, we typically use a total fluorescence imaging time of 100 seconds, e.g., 500 frames with 200 ms exposure time (Figure 4a), and an illumination power density on the order of 100–200 W/cm 2 , depending on the grid type 19 .…”

“…This issue has prompted the investigation of alternative super-resolution methods for imaging cryosamples 5,16 , and might in the future be avoided by the development of novel fluorescent probes that require less intense illumination to blink 17 . Currently, the way to avoid light-induced damage while using high illumination power densities is to select specific support grids that are capable of withstanding high illumination power densities, such as grids with low absorption support film, high mesh numbers, or grids with a low fraction of the area covered by absorbing material 18,19 .…”

Recent advances in infectious disease research using cryo-electron tomography

“… Yang et al (2023a) , developed a workflow for 3D correlative cryo-FLM-FIB-ET that precisely targets to ROIs with iFLM and cryo-FIB SEM. Pairing of super-resolution light microscopy and correlated electron microscopy is further being strengthened by optimal support grids ( Last et al, 2023 ). Recently, Xenon plasma FIB milling was used to make lamella directly from human brain tissue derived through biopsy to visualize sub-cellular features ( Creekmore et al, 2023 ).…”

Imaging intracellular componentsin situusing super-resolution cryo-correlative light and electron microscopy

“…The type of EM grid that is used is another important parameter in cryoSMLM, and dictates the maximum fluorescence excitation light intensity that can be used. We and others have previously reported on this topic, and we refer to those reports for more in-depth information 17,18 . Here, we use 1.2/1.3 UltrAuFoil 300 mesh holey gold grids, which are commercially available and well suited for cryoSMLM in general, and especially for adherent mammalian cells.…”