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High resolution correlative 3D imaging of the intracellular world under physiological conditions using the synergies of laser light and X-ray microscopes at cryogenic temperatures

High resolution correlative 3D imaging of the intracellular world under physiological conditions using the synergies of laser light and X-ray microscopes at cryogenic temperatures

Session

Stream 6 (Frontiers): Correlative Imaging of Organelle Organization and Architecture

Authors

Dr Chidinma Okolo (1), Dr Ilias Kounatidis (1), Dr Perrine Paul-Gilloteaux (2), Dr Ian Dobbie (3), Dr Maria Harkiolaki (1)

Affiliations

1. Diamond Light Source
2. Université de Nantes
3. University of Oxford

Keywords

soft X-ray tomography, fluorescence imaging, biological cryo-imaging, correlative microscopy, organelle localisation

Abstract text

Summary:

 

Correlative bioimaging of cell populations in 3D and under any number of experimental conditions can be achieved and stands to benefit from a new imaging platform that harnesses visible light structured illumination microscopy (SIM) and soft X-ray tomography (SXT) that is now available at the correlative cryo-imaging synchrotron beamline B24 at Diamond Light Source. The power of this method is demonstrated through projects that capture the way cells respond to environmental cues such as vesicle and membrane remodelling upon exposure to infectious agents.

 

Introduction:

 

Biological cryo-imaging has developed rapidly in recent years across scales, dimensions and resolutions. To capture the dynamic crosstalk of structures and processes within living cells, different imaging modalities need to complement each other perfectly. At the correlative cryo-imaging beamline, B24, at the UK synchrotron (Diamond Light Source), two high-resolution 3D imaging systems have been developed side-by-side to enable such in-depth examination of biological systems at near-physiological states. These two imaging methods are (1) fluorescence cryo-Structured Illumination Microscopy (cryoSIM) which highlights localisation of tagged molecules, organelles and other cellular structures within the cellular ultrastructure and (2) cryo soft X-ray Tomography (cryoSXT) which uses the natural absorption contrast of vitrified hydrated biological material to deliver high-resolution 3D data on biological systems and their cellular architecture (Kounatidis et al., 2020). The current fully commissioned workflow starts with samples first imaged in cryoSIM to generate 3D fluorescence information at high resolution and identify areas of interest before they are loaded to the transmission X-ray microscope for SXT data collection on these same areas of interest. This way, data recorded at different microscopes can be directly correlated enabling the unambiguous interpretation of data. In this fashion, we have succeeded in capturing snapshots of the cellular ultrastructure at distinct time points, post-trigger events in 3D and in multi-colour. Data will be presented from representative projects that demonstrate this correlative imaging scheme in action and these will be used to highlight the advantages, restrictions and critical points in our method.

 

 

Methods:

At Diamond Light Source beamline B24, a bespoke biological imaging platform has been developed offering SIM in conjunction with SXT. First, cells or other biological material such as vaccine formulations are cultured or deposited on the carbon film of gold grids. Following tissue culture, cells are exposed to biological or environmental/chemical cues or triggers. Thereafter, fluorescent trackers and nanoparticles are added prior to vitrification by plunge freezing at set time points. This is followed by mapping, 3D SIM imaging and reconstruction. Fluorescence imaging is followed by 2D X-ray mosaic acquisition and area-of-interest tilt series acquisition with X-rays; tilt series are then reconstructed into 3D tomographic volumes using IMOD. Multichannel 3D data generated through SIM and SXT data are correlated using ec-CLEM and are further subjected to morphometric analyses and segmented using Fiji. 

 

Results:

Recent correlative data at B24 have explored cell responses to external stimuli. The accumulated benefit of using (a) a non-invasive sample preparation strategy, (b) shuttling samples between microscopes without the need for further chemical or mechanical modifications and (c) the development of a bespoke user-friendly in silico data correlation process allows for quick and efficient data acquisition and analysis. The current imaging platform has enabled the unambiguous imaging of reovirus through the cellular machinery before the release of viral capsids to the cytoplasm. In the process, we have shown the dynamic relationship between the cytoskeleton, vesicles and membrane structures throughout the life of a cell. 

 

 

Conclusion:

 

Here, we present a unique and fully commissioned correlative microscopy platform that allows the investigation of cell populations at near-physiological condition and to tens of nanometer resolution using both laser and X-ray radiation. This way, it is providing researchers with both the context of cellular sub- and ultra-structure and the 3D localization of chemical information within it. The technology is now fully commissioned and accessible to everyone. 

References

Kounatidis, I., Stanifer, M. L., Phillips, M. A., Paul-Gilloteaux, P., Heiligenstein, X., Wang, H., Okolo, C. A., Fish, T. M., Spink, M. C., Stuart, D. I., Davis, I., Boulant, S., Grimes, J. M., Dobbie, I. M., & Harkiolaki, M. (2020). 3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway. Cell, 182(0), 1–16. https://doi.org/10.1016/j.cell.2020.05.051.