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- Chondrocyte (2)
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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.
The study was aimed at demonstrating a true cellular resolution for articular cartilage using synchrotron radiation-based X-ray microcomputed tomography (SR-µCT) with a sample-specific optimization of the phase contrast. The generated tomographic data were later used to prepare a matching histological sample from the full volume specimen.
We used highly coherent and monochromatic X-rays from a synchrotron source to image a tissue sample of bovine articular cartilage after deparaffinization. Phase contrast enhancement was achieved by using five different sample to detector distances for the same X-ray energy. After tomography, the sample was re-embedded into resin while retaining a dedicated sample orientation for subsequent sectioning and polishing, which was conducted until a previously defined spatial position was achieved. The protocol for resin embedding was developed to inhibit morphological changes during embedding. Giemsa staining was applied for better structural and morphological discrimination. Data from tomography and lightmicroscopy were exactly matched and finally compared to results from FIB/SEM imaging. Image detail was achieved at a single cell resolution. Image detail was achieved at a single cell resolution, which has been estimated to be 0.833 µm/voxel in the tomographic data.
SR-µCT with optimized phase contrast properties represents a method to investigate biological tissues in certain areas of interest, where true cellular resolution or enhanced volumetric imaging is needed. In this study, we demonstrate that this method can compete with conventional histology using light microscopy but even surpasses it due to the possibility of retrieving volumetric data.
Synchrotron radiation-based microcomputed tomography (SR-µCT) has become a valuable tool in the
structural characterization of different types of materials, achieving volumetric details with micrometre
resolution. Biomedical research dealing with porous polymeric biomaterials is one of the research fields
which can benefit greatly from the use of SR-µCT. This study demonstrates that current experimental setups
at synchrotron beamlines achieve a sufficiently high resolution in order to visualize the positions of
individual cartilage cells cultivated on porous gelatine scaffolds made by a freeze-structuring technique.
Depending on the processing parameters, the pore morphology of the scaffolds investigated was changed
from large-pore sized but non-ordered structures to highly directional and fine pored. The cell-seeded
scaffolds were stained with a combined Au/Ag stain to enhance the absorption contrast in SR-µCT. While
only some cells showed enhanced absorption contrast, most cells did not show any difference in contrast
to the surrounding scaffold and were consequently not detectable using conventional greyscale threshold
methods. Therefore, using an image-based three-dimensional segmentation tool on the tomographic data
revealed a multitude of non-stained cells. In addition, the SR-µCT data were compared with data obtained
from scanning electron microscopy, energy dispersive X-ray spectroscopy and histology, while further
linking the initial cell density measured via a MTT assay to the pore size as determined by SR-µCT.