Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells

Abstract

Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques.

High-resolution three-dimensional probes of biomaterials and their interfaces

“…Moreover, if we assume that high-resolution refers to the ability to resolve features on the nanometre scale, then, in fact, only electron and atom probe tomography meet this constraint. Even synchrotron source X-ray computed tomography, regularly employed in the characterization of biomaterials and their tissue interfaces, generally provides resolution in the micrometre range [7,8], albeit recent advances and specialized instrumentation have pushed the resolution into the nanometre regime [9]. Perhaps the highest resolution tomographic technique, atom probe tomography, enables atomic resolution in three dimensions.…”

Three-dimensional imaging of human stem cells using soft X-ray tomography

“…This in turn leads to noisy and artefact-corrupted reconstructed three-dimensional images. There are a number of studies reported where hard X-rays are used for tomographic image reconstruction that penetrate tens of micrometres into tissues and cell clusters, but these methods achieve much lower spatial resolution (micrometre range) than using the SXT method described here [48][49][50].…”

High Resolution X-Ray Tomography - 3D Imaging for Tissue Engineering Applications

“…Later on, this chapter deals with approaches using stacks of polymer sheets for possible applications in neuronal tissue engineering. Fig.2 has been used predominantly in this chapter and is described in several studies (Zehbe 1 et al, 2007;Zehbe 1 et al, 2009;Rack et al, 2008). Although the photon flux density is several orders of magnitude higher than in X-ray tubes, it is not constant over the X-ray energy.…”

“…While imaging in absorption contrast is well adapted to samples featuring a prominent difference in density or atomic mass, phase contrast imaging allows detection of structural details which are not well visible in absorption. Here, the contrast is achieved by retardation or refraction of coherent X-rays at phase boundaries inside the sample (Cloetens et al, 2006;Rack et al, 2008;Zehbe 1 et al, 2009). Fig.…”