What is a Confocal Microscope?

Confocal microscopy is an optical imaging technique that uses point-by-point scanning illumination and spatial pinhole filtering to remove out-of-plane scattered light from the focal point of the sample. Although the spatial resolution capability of this technology has not theoretically exceeded the diffraction limit, compared with traditional imaging, the object image contrast of this technology is significantly improved, thereby optimizing the image quality.

Confocal microscopy (Confocal microscopy) is an optical imaging method that uses point-by-point illumination and spatial pinhole modulation to remove scattered light from the non-focus plane of the sample. Visual contrast.

There are three types of commercial confocal microscopes:

• Confocal Laser Scanning Microscope (CLSM, LSCM)

• Spinning-disk (Nipkow disk) confocal microscopes

• Programmable Array Microscopes (PAM) ^[1]

  The probe light emitted from a point light source is focused by the lens onto the object under observation.

  • Confocal laser scanning microscopy ( CLSM , LCSM ) is a high-resolution three-dimensional optical imaging technology. The main feature is its optical layering ability, that is, to obtain an image in focus at a specific depth. The images were acquired point by point and then reconstructed by a computer. It can therefore reconstruct objects with complex topologies. For opaque samples, surface mapping can be performed, and for transparent samples, internal structure imaging can be performed. In internal structure imaging, the image quality can be greatly improved in a single microscope, because the information from different depths of the sample is not overlapped. Traditional microscopes can "see" everything that can be thrown into place by light, while for confocal microscopes, only the information at the focal point is collected. In fact, confocal laser scanning microscopy is achieved by controlling the depth of the focus and limiting the height.

    The principle of confocal was registered as a patent by American scientist Marvin Minsky as early as 1957, but in fact, after 30 years and the development of the corresponding special laser, this technology did not become a standard technology until the late 1980s. In 1978, Thomas and Christopher Kramer designed a laser scanning program. The program uses laser focusing to scan the three-dimensional surface of the object point by point, and generates images by computerized means similar to scanning electron microscope. This confocal laser scanning microscopy design combines the laser scanning method with the three-dimensional detection of fluorescently labeled biological samples for the first time. In the following decades, confocal fluorescence microscopy has developed into a mature technology, especially benefiting from the University of Amsterdam, the European Molecular Biology Laboratory from Heidelberg and its industry partners Work hard. ^[2]