Imaging and characterizing cells using tomography

Abstract

We can learn much about cell function by imaging and quantifying sub-cellular structures, especially if this is done non-destructively without altering said structures. Soft x-ray tomography (SXT) is a high-resolution imaging technique for visualizing cells and their interior structure in 3D. A tomogram of the cell, reconstructed from a series of 2D projection images, can be easily segmented and analyzed. SXT has a very high specimen throughput compared to other high-resolution structure imaging modalities; for example, tomographic data for reconstructing an entire eukaryotic cell is acquired in a matter of minutes. SXT visualizes cells without the need for chemical fixation, dehydration, or staining of the specimen. As a result, the SXT reconstructions are close representations of cells in their native state. SXT is applicable to most cell types. The deep penetration of soft x-rays allows cells, even mammalian cells, to be imaged without being sectioned. Image contrast in SXT is generated by the differential attenuation soft x-ray illumination as it passes through the specimen. Accordingly, each voxel in the tomographic reconstruction has a measured linear absorption coefficient (LAC) value. LAC values are quantitative and give rise to each sub-cellular component having a characteristic LAC profile, allowing organelles to be identified and segmented from the milieu of other cell contents. In this chapter, we describe the fundamentals of SXT imaging and how this technique can answer real world questions in the study of the nucleus. We also describe the development of correlative methods for the localization of specific molecules in a SXT reconstruction. The combination of fluorescence and SXT data acquired from the same specimen produces composite 3D images, rich with detailed information on the inner workings of cells.

Resolving new ultrastructural features of cytokinetic abscission with soft-X-ray cryo-tomography

“…Soft X-ray Cryo-Tomography (cryo-SXT) technique offers a complementary approach to electron and visible light based microscopy techniques121314. SXT takes advantage of the X-ray spectral window between atomic absorption transitions of carbon and oxygen, the so-called “water window”.…”

“…At these energies, carbon absorbs the illuminating radiation much more strongly than oxygen, so that carbon-rich structures such as proteins, membranes, and lipids appear dark on a relatively bright background from the aqueous medium. Cellular ultrastructure can then be characterized without addition of any stain or contrast agent, as image contrast represents a quantitative map of the local carbon density121315. Indeed, over the past few years, cryo-SXT has been demonstrated as a powerful tool for visualizing biological membranes and for determining the shape and size of membrane based organelles161718.…”

Quantitative imaging of Candida utilis and its organelles by soft X‐ray Nano‐CT

“…The absorption of soft X-rays obeys the Beer-Lambert law. The linear absorption coefficient (LAC) (Weiss et al, 2000) is a characteristic of the absorption capacity of substances (Do et al, 2015). Given the initial intensity of photons I 0 , the photon intensity I detected by the CCD is as follows (Weiss et al, 2000):…”

Mesoscale imaging with cryo‐light and X‐rays: Larger than molecular machines, smaller than a cell

“…For soft X-ray imaging, cells are simply taken from their growth media or tissue explant, mounted on a grid or in a capillary, and instantly cryo-preserved. The cells are imaged intact, fully hydrated, and unstained (Parkinson et al 2013, Do et al 2015). The resultant images do not contain artefacts due to procedures such as sectioning, chemical fixation or dehydration.…”