Cryo soft X-ray tomography using low-energy X-rays is a powerful method for imaging the 3D ultrastructure of whole cells to around 50 nm resolution. Cells or tissue blocks up to 10 µm thick are simply flash frozen and since the contrast is generated naturally, no staining or chemical fixatives are needed. This near-native state structural imaging modality provides volume information that is highly complementary to both electron microscopy and fluorescence microscopy-based techniques.
SiriusXT has developed a soft X-ray microscope (the SXT100) that is now routinely imaging a variety of mammalian and non-mammalian cryo-vitrified cell samples. The complete workflow is now in place to prepare and plunge freeze cells on standard 3 mm TEM grids, and perform grid inspection by transmitted light and fluorescent microscopy (FM), before transfer into the cryo-vacuum chamber of the soft X-ray microscope for tomographic imaging. The sample can be rotated through ±60° in 1° or 2° steps, with a projection image acquired at each angle. Standard alignment and reconstruction software used by the electron tomography community can be used to align and reconstruct the final image stack. The microscope is currently being validated through a series of real-world virology applications as part of the EU H2020 project CoCID. It is anticipated the microscope will be rolled out to general users when the company launches its ‘Imaging as a Service’ offering later in 2022.
Cryo soft X-ray tomography of whole cells and bulk tissue samples in the laboratory
Kenneth Fahy, Paul Sheridan, Tony McEnroe, et.al