Biology subjects

Sharp, T. H.

Publications and source records attributed to Sharp, T. H..

6 recordsLinked to original sources

bioRxiv2025

Scaling data analyses in cellular cryoET using comprehensive segmentation

Automation and improved hardware have greatly accelerated the rate of data generation in cryoET. As the field moves towards quantitative cryoET, the scale of the resulting datasets presents a significant challenge for analysis and interpretation. To explore ways of handling datasets comprising thousands of tomograms, we investigated a comprehensive segmentation strategy - assigning an ontology-based identity to every voxel in a dataset - that is based on the sequential application of multiple convolutional neural networks. Using an openly available dataset of over 1800 Chlamydomonas reinhardtii tomograms as a test case, we demonstrate the segmentation of 25 different subcellular features across the full dataset, while requiring only a few seconds of processing time per tomogram. We show how the approach enables the representation of large datasets as searchable databases and propose the usage of ontology-based segmentations for improving two common processing tasks in cryoET. First, we explore context-aware particle picking as a method to retain biological context when selecting particles for subtomogram averaging and other downstream analyses. Secondly, we demonstrate area-selective template matching, where we use segmentation-based masks to avoid redundant computations in template matching and enable >500-fold faster processing in specific cases. To illustrate the utility of the approach, all segmentation results have also been made available online via cryopom.streamlit.app.

molecular biology↗

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

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↗

bioRxiv2022

Plasmodium falciparum has evolved multiple mechanisms to hijack human immunoglobulin M

Plasmodium falciparum causes the most severe malaria in humans. Immunoglobulin M (IgM) serves as the first line of humoral defense against infection and potently activates the complement pathway to facilitate P. falciparum clearance. A number of P. falciparum proteins hijack IgM, leading to immune evasion and severe disease. However, the underlying molecular mechanisms remain unknown. Here, using high-resolution cryo-electron microscopy, we delineate how P. falciparum proteins VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2 target IgM. Each protein binds IgM in a different manner, and together they present a variety of Duffy-binding-like domain-IgM interaction modes. We further show that these proteins interfere directly with IgM-mediated complement activation, with VAR2CSA exhibiting the most potent inhibitory effect. Structural analyses suggest that VAR2CSA occludes the congregation of the complement C1 complex on IgM. These results underscore the importance of IgM for the adaptation of P. falciparum to humans, and provide critical insights into the immune evasion mechanism of P. falciparum.

biochemistry↗