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Background <p>Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300&#xa0;kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100&#xa0;kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200&#xa0;kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications.</p> Methods <p>Here, we set out to investigate if a 200&#xa0;kV electron cryomicroscope without an energy filter can be utilized for <i>in situ</i> structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets.</p> Results <p>We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ~ 15&#xa0;Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200&#xa0;kV instruments are heavily underutilized with respect to electron cryotomography applications.</p> Discussion <p>This study demonstrates that 200&#xa0;kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200&#xa0;kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.</p>

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In situ structural analysis of mammalian cells using a 200 kV electron cryomicroscope – implications for research infrastructure

  • 期刊论文

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  • 发表日期：

    2025年11月6日

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  • Piotr Szwedziak

摘要

Background

Electron cryotomography is a powerful imaging technique allowing for studying functional cellular modules in their native environment with macromolecular resolution. However, it requires access to complex and expensive instrumentation, typically a 300 kV electron cryomicroscope equipped with an energy filter. Simpler and cheaper 200 and 100 kV instruments have been successfully used for single particle cryoEM analyses, which has helped to democratize the technique and broaden access. It has not been systematically studied if 200 kV electron cryomicroscopes can deliver meaningful and interpretable data with respect to electron cryotomography applications.

Methods

Here, we set out to investigate if a 200 kV electron cryomicroscope without an energy filter can be utilized for in situ structural studies of mammalian cells by electron cryotomography of thin cell edges followed by extensive image analysis including segmentations, subtomogram averaging and molecular sociology studies of lipid droplets.

Results

We demonstrate that the resulting tomograms of thin edges of U2OS cells are of sufficient quality to annotate the contents of the cell and observe spatial inter-relationships among macromolecules. In particular, we undertook a molecular sociology analysis of lipid droplets and addressed their subcellular distribution and interactions with other organelles. Additionally, we performed subtomogram averaging of purified 70S ribosomes that resulted in ~ 15 Å resolution 3D reconstruction. Finally, we examined geographical distribution and scientific output of the two most common electron cryomicroscopy platforms and deduced that 200 kV instruments are heavily underutilized with respect to electron cryotomography applications.

Discussion

This study demonstrates that 200 kV electron cryomicroscopes can be utilized for structural cell biology studies by electron cryotomography. Given the favorable ratio of their versatility versus costs we foresee that 200 kV electron cryomicroscopes will become workhorses of local electron cryomicroscopy facilities.