Confocal laser scanning microscopy

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Confocal laser scanning microscopy (CLSM or LSCM) is a technique for obtaining high-resolution optical images with depth selectivity.[1] 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,[2] but it took another thirty years and the development of lasers for CLSM to become a standard technique toward the end of the 1980s.[1] In 1978, Thomas and Christoph Cremer designed a laser scanning process, which scans the three dimensional surface of an object point-by-point by means of a focused laser beam, and creates the over-all picture by electronic means similar to those used in scanning electron microscopes.[3] This CLSM design combined the laser scanning method with the 3D detection of biological objects labeled with fluorescent markers for the first time. During the next decade, confocal fluorescence microscopy was developed into a fully mature technology, in particular by groups working at the University of Amsterdam and the European Molecular Biology Laboratory (EMBL) in Heidelberg and their industry partners.

Contents

Image formation

In a confocal laser scanning microscope, a laser beam passes through a light source aperture and then is focused by an objective lens into a small (ideally diffraction limited) focal volume within or on the surface of a specimen. In biological applications especially, the specimen may be fluorescent. Scattered and reflected laser light as well as any fluorescent light from the illuminated spot is then re-collected by the objective lens. A beam splitter separates off some portion of the light into the detection apparatus, which in fluorescence confocal microscopy will also have a filter that selectively passes the fluorescent wavelengths while blocking the original excitation wavelength. After passing a pinhole, the light intensity is detected by a photodetection device (usually a photomultiplier tube (PMT) or avalanche photodiode), transforming the light signal into an electrical one that is recorded by a computer.[4]

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