A protocol for full-rotation soft X-ray tomography of single cells

Chen, Jianhua; Vanslembrouck, Bieke; Loconte, Valentina; Ekman, Axel; Cortese, Mirko; Bartenschlager, Ralf; McDermott, Gerry; Larabell, Carolyn A.; Gros, Mark A. Le; Weinhardt, Venera

doi:10.1016/j.xpro.2022.101176

Cited by 25 publications

(32 citation statements)

References 22 publications

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“…The diameter of the capillary is variable and based on cell size (scale bar, 8 μm) 39 . Each capillary is plunge‐frozen in liquid nitrogen‐cooled liquid propane using a custom‐made freezing apparatus, then stored in liquid nitrogen until data collection 22,28 . (B) Orthoslice of a human lymphoblastoid cell, with LAC values shown between 0.1–0.6 μm −1 ; the average values for each organelle are reported in brackets.…”

Section: Imaging Cells Using Soft X‐ray Tomographymentioning

confidence: 99%

“…Unique to SXT, the contrast of structures is quantitative, reflecting information about the molecular composition of each region of the cell. SXT imaging is also fast in that a tomographic reconstruction of a large eukaryotic cell can be generated in 5–10 min from mounting the specimen on the microscope, enabling analysis of statistically significant numbers of cells 28,29,35 . SXT data are useful for the development of cell models.…”

Section: Imaging Cells Using Soft X‐ray Tomographymentioning

confidence: 99%

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Soft X‐ray tomograms provide a structural basis for whole‐cell modeling

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Developing in silico models that accurately reflect a whole, functional cell is an ongoing challenge in biology. Current efforts bring together mathematical models, probabilistic models, visual representations, and data to create a multi‐scale description of cellular processes. A realistic whole‐cell model requires imaging data since it provides spatial constraints and other critical cellular characteristics that are still impossible to obtain by calculation alone. This review introduces Soft X‐ray Tomography (SXT) as a powerful imaging technique to visualize and quantify the mesoscopic (~25 nm spatial scale) organelle landscape in whole cells. SXT generates three‐dimensional reconstructions of cellular ultrastructure and provides a measured structural framework for whole‐cell modeling. Combining SXT with data from disparate technologies at varying spatial resolutions provides further biochemical details and constraints for modeling cellular mechanisms. We conclude, based on the results discussed here, that SXT provides a foundational dataset for a broad spectrum of whole‐cell modeling experiments.

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Section: Imaging Cells Using Soft X‐ray Tomographymentioning

confidence: 99%

Section: Imaging Cells Using Soft X‐ray Tomographymentioning

confidence: 99%

Soft X‐ray tomograms provide a structural basis for whole‐cell modeling

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“…The cells were loaded into thin-wall glass capillaries with a micro-loader. The capillaries were rapidly plunged into a liquid nitrogen cooled liquid propane and stored in liquid nitrogen until image acquisition 64 .…”

Section: Freezing Of Cellsmentioning

confidence: 99%

Extending of imaging volume in soft x-ray tomography

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Chen

Vanslembrouck

et al. 2022

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Soft x-ray tomography offers rapid whole single cell imaging with a few tens of nanometers spatial resolution without fixation or labelling. At the moment, this technique is limited to 10 µm thick specimens, such that applications of soft x-ray tomography to large human cells or multicellular specimens are not possible. We have developed a theoretical and experimental framework for soft x-ray tomography to enable extension of imaging volume to 18 µm thick specimens. This approach, based on long depth of field and half-acquisition tomography, is easily applicable to existing full-rotation based microscopes. This opens applications for imaging of large human cells, which are often observed in cancer research and cell to cell interactions.

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“…Lipids were based on DOPC (C40H80NO8P), with MAC value of 9264.835/cm and a volume of 1150.0 Å 3 per lipid (Greenwood et al 2006), and a value of 1114.279/cm was used for water.To generate the final images, the initial calculated volume is embedded in a larger volume the size of the experimental data. The simulated reconstructions were obtained similar to the experimental ones: the projection images were distorted by Poisson noise corresponding to the shot noise of the experiment, and random translations were added to the tomograms to mimic sub-pixel alignment errors of the image registration(Chen et al 2022). The reconstruction was repeated 10 times for each volume and averaged for calculation of the radial profiles.…”

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Integrative structural modelling and visualisation of a cellular organelle

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Models of insulin secretory vesicles from pancreatic beta cells have been created using the cellPACK suite of tools to research, curate, construct and visualise the current state of knowledge. The model integrates experimental information from proteomics, structural biology, cryoelectron microscopy and X-ray tomography, and is used to generate models of mature and immature vesicles. A new method was developed to generate a confidence score that reconciles inconsistencies between three available proteomes using expert annotations of cellular localisation. The models are used to simulate soft X-ray tomograms, allowing quantification of features that are observed in experimental tomograms, and in turn, allowing interpretation of X-ray tomograms at the molecular level.

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A protocol for full-rotation soft X-ray tomography of single cells

Cited by 25 publications

References 22 publications

Soft X‐ray tomograms provide a structural basis for whole‐cell modeling

Soft X‐ray tomograms provide a structural basis for whole‐cell modeling

Extending of imaging volume in soft x-ray tomography

Integrative structural modelling and visualisation of a cellular organelle

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