XRM2024 - Thu08A - "Correlative cryo soft x-ray tomography and fluorescent microscopy of biological samples in the laboratory"

Soft X-ray tomography (SXT) uses low energy X-rays to image frozen-hydrated biological specimens such as entire mammalian cells or thick tissue slabs with a few tens of nanometers of spatial resolution [1-4]. The technique takes advantage of the biological water window whereby water is relatively transparent to X-rays from 284 to 543 eV (2.34 to 4.4 nm) but carbon-based organelles are absorbing. This native contrast imaging allows high resolution imaging of bulk specimens with minimal sample preparation. While SXT has traditionally been confined to synchrotrons the recent development of a laboratory scale SXT microscope opens the possibility of integrating this novel technique into light and electron imaging workflows [5]. The SXT microscope features an integrated light microscope for overview imaging and fluorescence targeting, allows for swift acquisition of 2D and 3D X-ray images covering extensive areas on the specimen, and enables efficient and rapid identification of cells of interest. The utility of the microscope is currently being demonstrated through 2 EU-funded consortia (https://cocid.eu/ and https://clexm.eu/) [6]. Recent case studies have included correlative imaging of whole, virus infected cells treated with anti-viral drugs; imaging the 3D distribution of therapeutic-laden nanoparticles in whole cells; and the development of correlative workflows for tissue imaging across scales [7-9]. An alternative sample carrier in the form of a thin-walled glass capillary was also developed, further enhancing the novelty of the lab-based microscope.

Integrated cryo fluorescence was used to screen an entire EM grid for cell selection and correlation with SXT. Low magnification/large field of view 2D X-ray mosaics were then acquired over large areas of the grid before acquiring tilt series from ±60° on selected targets. Data from both modalities were then overlaid to provide the location of fluorescent proteins in the context of whole cell ultrastructure. For cells suspended in glass capillaries the tilt series was acquired over ±90°. Resulting cryo-SXT tomograms and fluorescent light images of cellular organelles and nanoparticles provide proof of concept for the lab-based microscope and associated workflows.

Workflows of correlative light, electron and soft X-ray microscopy combines the strengths of each modality. The recent availability of a compact soft X-ray microscope will accelerate the development of novel workflows and biological imaging applications that can benefit from this technique. We will present our microscope workflows and application data.

References:

[1] Guo, J. and Larabell, C.A., (2019). Current Opinion in Structural Biology 58, 324
[2] Schneider, G. et al., (2010). Nat. Methods 7, 985
[3] Harkiolaki, M. et al., (2018). Emerging Topics in Life Sciences 2, 81
[4] Chichón, F.J. et al., (2012). Journal of Structural Biology 177, 202
[5] Fahy, K. et al., (2024). Methods in Cell Biology, Academic Press, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2024.02.033.
[6] The authors acknowledge funding from the European Union’s Horizon 2020 Research and Innovation programme (No. 101120151, project CLEXM and No. 101017116, project CoCID) as well as the Irish Research Council (No. EBPPG-2020-278).
[7] Castro, V. et al., (2025). Structure 33(9), 1547
[8] O’Connor, S., Rogers, D. et al., (2025). Sci Rep 15, 45491
[9] Groen, J. et al., (2025). Commun Biol 8, 1677