Cryo‐Soft X‐ray Tomography of the Cell

Abstract

Abstract Cryo soft X‐ray tomography (cryo‐SXT) is an emerging X‐ray microscopy technique for three‐dimensional visualisation of cryo‐preserved, whole, unstained cells at a spatial resolution of 30 nm (half‐pitch). It is, therefore, bridging the gap between the low‐resolution visible light imaging techniques that provide dynamic information of specific processes, and the high resolution electron microscopy ones which achieve molecular resolution. As a first‐order approximation, in SXT as in electron tomography, a series of absorption contrast projections of the specimen are collected at different angles to finally compute the 3D absorption maps. In addition to the 3D structural information that can be obtained, the chemical sensitivity of the X‐rays to the atomic elements of the sample can also be exploited to enhance visualisation of these elements or to distinguish different chemical compositions, as well as to extract quantitative information from the data. Key Concepts: Tomography allows three‐dimensional visualisation of the sample. The soft X‐ray water window energy range allows penetrating up to 10 μm of water therefore enabling a full cell to be imaged. Cryo‐fixation is the only sample preparation step required in soft X‐ray tomography (SXT). Cryo soft X‐ray tomography data suffers from the limited depth of field of the Fresnel zone plate objective lens which is usually smaller than the thickness of vitreous ice that can be penetrated. The interpretation of soft X‐ray tomography data is complex and requires complementary information from electron and optical microscopy.