Cryo-soft X-ray tomography: using soft X-rays to explore the ultrastructure of whole cells

Abstract

Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.

X-ray computed tomography in life sciences

“…The limited penetration of soft Xrays means soft nCT is limited to a sample thickness of 10 μm [74], whereas samples many tens of microns in diameter can be imaged using hard nCT at a spatial resolution down to 50 nm [15]. This provides the possibility of imaging cells within native tissues or when seeded onto biomaterial scaffolds, whereas soft nCT has typically been used to image adherent cells cultured on 2D surfaces [75], or cells in suspension [61]. For example, hard nCT has been used to observe human femur over a field of view of 9 μm containing 17 lacunae, at a voxel size of 60 nm, in which collagen fibre orientation within the bone matrix could also be identified [76].…”

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway

“…For 3D EM visualization of thicker samples, such as mammalian cells that can easily grow beyond 3 μm in thickness, sectioning and serial reconstruction methods can be employed to overcome this limitation, but they involve chemical treatment of samples or complex and laborious cryogenic workflows ( Rigort and Plitzko, 2015 ; Son et al., 2013 ). Another high-resolution 3D imaging method, soft X-ray tomography (SXT), has to date filled this need for direct mesoscale imaging of cellular ultrastructure in thicker vitrified samples, offering a penetration depth in the order of 10 μm, to a resolution of a few tens of nanometers, and the added benefit that samples require no chemical processing ( Harkiolaki et al., 2018 ; Schneider et al., 2010 ). In addition, and complementary to both EM and SXT, optical microscopy continues to provide direct access to information on ultrastructure, organelle organization, and molecular localization within cells at variable resolution ranges ( Giepmans et al., 2006 ).…”

Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing

“…The SFF method should also find applications in the cryofixation in cellular imaging such as soft X-ray tomography, high-aperture cryolight microscopy, and their combination (37). Cryofixation by plunge freezing is widely used for the sample preparation in these imaging techniques, since it can halt all motion and metabolic activity (38). As far as the resolution is concerned, these imaging methods are not affected by the presence of small ice crystals that might be generated by the SFF method.…”