Therefore, sensitive detectors such as photomultiplier tubes or avalanche photodiodes are normally used in order to limit the required exposure time. The obtained optical power registered by the photodetector is usually rather small. It is essential to ensure that the pinhole is adjusted such that one collects light exactly from the laser beam focus. In addition, the laser intensity quickly gets smaller due to the substantial beam divergence. Light from positions in the sample above or below the beam focus can also not well get through the pinhole because its focus position is not in the plane of the pinhole (the selected optically conjugate plane).Also, such light could not get through the pinhole, since it would be focused to points away from the hole. At other lateral positions, there is hardly any incident laser light, except somewhat before and after the beam focus due to the substantial beam divergence.Light coming from other longitudinal or transverse positions in the sample is largely suppressed: with oscillating mirrors) or alternatively the sample, scanning a certain area or volume in the sample ( flying spot microscopy). A complete two-dimensional or three-dimensional image is obtained by systematically translating either the beam focus (with some kind of laser scanner, e.g. The recorded intensity conveys information on just a single object point.through scattering in the sample, or fluorescence light) is imaged to a small pinhole and transmitted to a photodetector. The light coming back from the focus point (e.g.A diffraction-limited collimated laser beam is tightly focused onto the sample, using a microscope objective ( point illumination instead of illumination of the whole object).(The confocal principle was actually recognized even earlier, but apparently the term has been introduced only much later, in 1977, in the context of a thorough theoretical analysis of the operation principle of confocal microscopy.) Figure 1: Setup of a confocal laser microscope.Ī confocal scanning microscope (see Figure 1) in a modern form essentially works as follows: Substantially enhanced longitudinal (axial) resolution can be obtained by utilizing the principle of confocal microscopy, which was invented and patented by Marvin Lee Minsky in the 1950s, i.e., before the invention of the laser. Some limited amount of suppression is obtained by the fact that contributions from light outside the object plane (selected with the focus adjustment) are out of focus, i.e., strongly blurred. A remaining problem, however, is that light from different longitudinal positions in a transparent sample can contribute to an image, leading to a limited longitudinal resolution. In a conventional optical microscope, a quite high lateral image resolution (well below 1 μm) can be achieved by using a microscope objective with short focal length and a high numerical aperture. Operation Principle of Confocal Microscopy The principle of confocal microscopy is also applied in some optical profilometers. Therefore, such instruments have become important for biological and medical research and various other areas of research and industrial inspection. With such microscopes, one can even observe tiny details within living cells, for example. of transparent biological and medical samples – effectively applying a kind of “optical sectioning” instead of physical sectioning of samples. This allows one to acquire three-dimensional images e.g. How to cite the article suggest additional literatureĬonfocal microscopes are scanning microscopes, the primary feature of which is a substantially improved longitudinal (axial) resolution by suppressing image contributions from light outside a rather thin plane. Acronym: CLSM = confocal laser scanning microscopeĭefinition: optical microscopes with enhanced depth resolution based on the confocal measurement principleĬategories: vision, displays and imaging, optical metrology
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