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Laser Scanning Confocal Microscope
source:     author:     2009-10-24

  

  Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects. For opaque specimens, this is useful for surface profiling, while for non-opaque specimens, interior structures can be imaged. For interior imaging, the quality of the image is greatly enhanced over simple microscopy because image information from multiple depths in the specimen is not superimposed. A conventional microscope "sees" as far into the specimen as the light can penetrate, while a confocal microscope only images one depth level at a time. In effect, the CLSM achieves a controlled and highly limited depth of focus. The principle of confocal microscopy was originally patented by Marvin Minsky in 1957, but it took another thirty years and the development of lasers for CLSM to become a standard technique toward the end of the 1980s. Thomas and Christoph Cremer designed in 1978 a laser scanning process which scans point-by-point the three dimensional surface of an object by means of a focused laser beam and creates the over-all picture by electronic means similar to those used in scanning electron microscopes. It is this plan for the construction of a CSLM, which for the first time combined the laser scanning method with the 3D detection of biological objects labeled with fluorescent markers. During the next decade, confocal fluoresence microscopy was developed into a technically fully matured state in particular by groups working at the University of Amsterdam and the European Molecular Biology Laboratory (EMBL) in Heidelberg and their industry partners.

  CLSM is widely-used in numerous biological science disciplines, from cell biology and genetics to microbiology and developmental biology.

  Clinically, CLSM is used in the evaluation of various eye diseases, and is particularly useful for imaging, qualitative analysis, and quantification of endothelial cells of the cornea. It is used for localizing and identifying the presence of filamentary fungal elements in the corneal stroma in cases of keratomycosis, enabling rapid diagnosis and thereby early institution of definitive therapy. Research into CLSM techniques for endoscopic procedures is also showing promise. In the pharmaceutical industry, it was recommended to follow the manufacturing process of thin film pharmaceutical forms, to control the quality and uniformity of the drug distribution. CLSM is also used as the data retrieval mechanism in some 3D optical data storage systems and has helped determine the age of the Magdalen papyrus.  


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