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 (FM) 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, and demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated microscopy.

Super-resolution fluorescence imaging of cryosamples does not limit achievable resolution in cryoEM

“…After plunge freezing, samples were irradiated in a custom-built fluorescence cryo-microscope 2 that is equipped with high powered 405, 488, and 561 nm lasers (100-150 mW, LightHUB, Omicron-Laserage GmbH, Rodgau, Germany). The illumination power density was determined using a S130C power meter (Thorlabs, Newton, NJ, USA) to measure the light output in the focal plane at the front aperture of the objective lens and dividing that value by the size of the illuminated spot, as measured directly from images.…”

“…Samples were illuminated in a custom-built fluorescence cryo-microscope that we have previously used to determine safe illumination power densities for fluorescence microscopy 2 . Illumination power densities were limited in order to avoid devitrification or melting, yet high enough (100-140 W/cm 2 ) to be suitable for single-molecule localization microscopy 2,[10][11][12] . With the knowledge that these photon dose rates are sufficiently low to maintain sample integrity, we now wanted to investigate the effect of the total photon dose.…”

“…Recent reports demonstrate that by avoiding optical heating of samples in correlated fluorescence and transmission electron cryo-microscopy, high illumination power densities can be used that enable better super-resolution fluorescence imaging 1,2 . While this rules out (which was not certified by peer review) is the author/funder.…”

“…Recent reports demonstrate that by avoiding optical heating of samples in correlated fluorescence and transmission electron cryo-microscopy, high illumination power densities can be used that enable better super-resolution fluorescence imaging 1,2 . While this rules out devitrification as a mode of damaging the sample, it is not clear whether fluorescence inspection of a sample prior to cryoEM imaging damages the sample via other possible mechanisms, such as via the illumination of highly absorptive fluorescent proteins (FPs) with intense laser light.…”

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.…”