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.
Highlights
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 or highresolution imaging of very thin samples
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
One half was used for histological characterization using light microscopy, while the other half was used for the Synchrotron radiation-based micro-computed tomography (SR-mCT) analysis and Scanning electron microscopy (SEM)/focused ion beam (FIB)
Summary
Most influenced by Morgagni (1761), Bichat (1802) and Virchow (1859) cumulating in the development of modern histology as one of the principal methodologies for the understanding of the structural organization of tissue in health, disease and regeneration. Bichat achieved considerable knowledge without using microscope equipment, since that time, microscopes in combination with tissue-specific staining protocols. This journal is q 2009 The Royal Society. Virchow (1859) established a concept that is focused on the cellular level of living processes and stated that all physiological disorders are based on morphological changes in organs, tissues and cells. Further efforts have been made in the optical components of light microscopes and in histological techniques. Samples that are larger can only be investigated using serial sectioning, a technique which is both time consuming and which requires precise alignment of probably deformed subsequent serial sections (Braverman & Braverman 1986; Bussolati et al 2005), while destroying the specimen
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