Correlative cryo-soft X-ray tomography of cells

摘要

Structural cell biology demands detailed structural and functional descriptions of the different cellular components which must be correlated with a topological 3D map of these components at the whole cellular level. Among the imaging techniques available, visible light microscopy has a privileged position due to its availability at laboratories worldwide and the possibility to image live.However, only the tagged features are being visualized, therefore producing a limited picture of the system. On the other hand, transmission electron microscopy (TEM) allows cell visualization at a higher spatial resolution, thus revealing ultrastructural cellular details. But it requires cellular sectioning as the multiple scattering of electrons imposes a restrictive sample thickness limitation. Complex protocols are therefore required for sample preparation; and overall, whole-cell visualization by TEMis complicated as stacked 2D sections imply difficult and time-consuming experiments. Even though the abovementioned visible light and electron microscopy techniques are broadly used, the correlation of information they provide is hindered firstly by the existing resolution gap in between them and secondly by the different sample thickness range each of them can handle. In this frame, an emerging technique such as cryo soft X-ray tomography (cryo-SXT) (Schneider et al., 2010) can provide structural information at the level of a whole cell without further sample preparation except for the cryo-fixation required to prevent radiation damage while collecting the data. The penetration power of soft X-rays in the so-called water window spectral range, between the inner-shell absorption edges of carbon and oxygen (from 284 to 543 eV), allows penetrating water layers of up to 10 μm thickness while carbon-rich structures are visualized with good absorption contrast (Wolter, 1952). Thus, frozen-hydrated specimens can be imaged close to their native state providing significant complementary