Jian-Hua Chen, Bieke Vanslembrouck, Valentina Loconte, Axel Ekman, Mirko Cortese, Ralf Bartenschlager, Gerry McDermott, Carolyn A. Larabell, Mark A. Le Gros, Venera Weinhardt,
A protocol for full-rotation soft X-ray tomography of single cells, STAR Protocols, Volume 3, Issue 1 2022,
The protocol described in this paper shows step-by-step sample preparation, data acquisition, and segmentation of cellular organelles with soft X-ray tomography. It is designed for microscopes built to perform full-rotation data acquisition on specimens in cylindrical sample holders, such as the XM-2 microscope at the Advanced Light Source, LBNL; however, it might be generalized for similar sample holder designs for both synchrotron and table-top microscopes.
The specimens are held in cylindrical sample holders which can be rotated through 360°, which eliminates artifacts caused by the limited tilt tomography of flat sample holders.
SXT of single cells allows the visualisation of whole-cell morphology, including the nucleus and chromatin distribution, mitochondria, lipid droplets, endoplasmic reticulum, endosomes, lysosomes, and intracellular vesicles.
This provides information about organelle spatial distribution and changes in the cell ultrastructure organization caused by a viral infection or other conditions, as well as by the alteration in the biochemical composition. The acquisition of multiple tomograms from the same cell type and cell condition allows the microscopist to map and quantify the mutual arrangement of the subcellular components, thus providing an average cellular phenotype.