Soft X-ray tomography (SXT) is one of the most recent structural biology techniques for quantitative three-dimensional (3D) analysis of whole cells.  When preserved in cryo-conditions, whole cells can be imaged by Fresnel zone plate (FZP) lenses with spatial resolution in the few tens of nanometers. Information from these images can then be combined by tomographic techniques to generate 3D maps of the specimen.  X-ray photon-energies in the so-called water window (between 284 and 543 eV) are especially useful, since high contrast images of cryopreserved whole cells of ~10 microns can be obtained without use of staining agents. However, SXT is far from currently being delivering its promise to its full extend, mostly due to the intrinsic limited depth of field (DOF) of the microscope optical system and to the over simplicity of the standard reconstruction algorithms being used. Indeed, this limited DOF produces images where the contribution of each plane of the specimen is different, leading to three-dimensional (3D) maps where certain parts of the specimen are blurred. We analyze the SXT image formation process in detail, providing a new mathematical theory allowing for the quantitative SXT inversion using focal series projections, images acquired at different defocus (Figure 1). Following these ideas, the SXT microscope at the Spanish synchrotron ALBA has been modified so as to automatically acquire the necessary additional information in a fast and reliable manner. Examples of the use of the new formulation on several biological systems using images acquired at ALBA are provided.

Here we apply our quantitative approach to SXT inversion to the study of an experimental biological system: a rotavirus infected cell tomogram (Figure 2).

Figures:

Figure 1. Acquisition scheme of focal series projections. Three tomograms are acquired at three different focal series projections. Each tomogram focuses on a different part of the specimen.

Figure 2. Comparison on experimental tomography of rotavirus infected cells. Microscope DOF = 2 μm and 31 nm half pitch resolution: 0-shift (first row) and XTEND (second row) reconstructions; (a,e) x-z planes where x-y slices at position -1.3 (b, f), 0 (c, g) and 1.5 μm (d, h) are marked. Scale bars = 1 μm

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EMC Abstracts - https://emc-proceedings.com/abstract/soft-x-ray-tomography-enhancement-by-focal-series-projections/