Principles

In the Confocal/Laser Scanning Microscopy, a laser light beam is expanded to make optimal use of the optics in the objective. Through a x-y deflection mechanism this beam is turned into a scanning beam, focused to a small spot by an objective lens onto a fluorescent specimen. The mixture of reflected light and emitted fluorescent light is captured by the same objective and (after conversion into a static beam by the x-y scanner device) is focused onto a photo-detector (photomultiplier) via a dichroic mirror (beam splitter). The reflected light is deviated by the dichroic mirror while the emitted fluorescent light passes through in the direction of the photomultiplier. A confocal aperture (pinhole) is placed in front of the photo-detector, such that the fluorescent light from the specimen that is not within the focal plane  where the laser beam was focused will be largely obstructed by the pinhole. In this way, out-of-focus information (both above and below the focal plane) is greatly reduced.

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