The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.