Biology subjects

Last, M. G. F.

Publications and source records attributed to Last, M. G. F..

5 recordsLinked to original sources

bioRxiv2024

Ais: streamlining segmentation of cryo-electron tomography datasets

Segmentation is a critical data processing step in many applications of cryo-electron tomography. Downstream analyses, such as subtomogram averaging, are often based on segmentation results, and are thus critically dependent on the availability of open-source software for accurate as well as high-throughput tomogram segmentation. There is a need for more user-friendly, flexible and comprehensive segmentation software that offers an insightful overview of all steps involved in preparing automated segmentations. Here, we present Ais: a dedicated tomogram segmentation package that is geared towards both high performance and accessibility, available at github.com/bionanopatterning/Ais. In this report, we demonstrate two common processing steps that can be greatly accelerated with Ais: particle picking for subtomogram averaging, and generating many-feature segmentations of cellular architecture based on in situ tomography data. Featuring comprehensive annotation, segmentation, and rendering functionality, as well as an open repository for trained models at aiscryoet.org, we hope that Ais will help accelerate research and dissemination of data involving cryoET.

molecular biology↗

bioRxiv2023

Imaging intracellular components in situ using super-resolution cryo-correlative light and electron microscopy

Super-resolution cryo-correlative light and electron microscopy (SRcryoCLEM) is emerging as a powerful method to enable targeted in situ structural studies of biological samples. By combining the high specificity and localization accuracy of single-molecule localization microscopy (cryoSMLM) with the high resolution of cryo-electron tomography (cryoET), this method enables accurately targeted data acquisition and the observation and identification of biomolecules within their natural cellular context. Despite its potential, the adaptation of SRcryoCLEM has been hindered by the need for specialized equipment and expertise. In this chapter, we outline a workflow for cryoSMLM and cryoET-based SRcryoCLEM, and we demonstrate that, given the right tools, it is possible to incorporate cryoSMLM into an established cryoET workflow. Using Vimentin as an exemplary target of interest, we exemplify all stages of an SRcryoCLEM experiment: performing cryoSMLM, targeting cryoET acquisition based single-molecule localization maps, and correlation of cryoSMLM and cryoET datasets using scNodes, a software package dedicated to SRcryoCLEM. By showing how SRcryoCLEM enables the imaging of specific intracellular components in situ, we hope to facilitate the further adaptation of the technique within the field of cryoEM.

molecular biology↗

bioRxiv2023

Building a super-resolution fluorescence cryomicroscope

Correlating super-resolution fluorescence microscopy with cryo-electron tomography is a recent advancement in the field of cryo-electron microscopy that enables targeted, high-resolution imaging of specific biomolecules of interest. Critical to this approach is that the cryo-correlated light and electron microscopy (cryoCLEM) workflow requires samples to be cryogenically fixed prior to imaging, and thus a fluorescence microscope is required that can maintain the cryogenically preserved state of the sample while also being capable of super-resolution imaging. In this report, we outline the blueprint of a microscope that was designed for single molecule localization microscopy of cryosamples, and we describe the rationale behind its design. All specifications, including a detailed 3d model of the entire assembly, are freely available via ccb.lumc.nl/downloads-231.

molecular biology↗

bioRxiv2023

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

Correlated super-resolution cryo-fluorescence and cryo-electron microscopy (cryoEM) has been gaining popularity as a method to investigate biological samples with high resolution and specificity. A concern in this combined method (called SR-cryoCLEM), however, is whether and how fluorescence imaging prior to cryoEM acquisition is detrimental to sample integrity. In this report, we investigated the effect of high-dose laser light irradiation on apoferritin samples prepared for cryoEM with excitation wavelengths commonly used in fluorescence microscopy, and comparing these samples to controls that were kept in the dark. We found that laser illumination, of equal duration and intensity as used in super-resolution cryomicroscopy and in the presence of high concentrations of fluorescent protein, did not affect the achievable resolution in cryoEM, with final reconstructions reaching resolutions of ~1.8 [A] regardless of the illumination conditions. The finding that super-resolution fluorescence imaging of cryosamples prior to cryoEM data acquisition does not limit the achievable resolution suggests that super-resolution cryo-fluorescence microscopy and in situ structural biology using cryoEM are entirely compatible. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2022

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

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.

molecular biology↗