Laser scanning microscope
A laser scanning microscope (rare laser scanning microscope; English laser scanning microscope , LSM , and scanning laser microscope ) is a light microscope in which a focused laser beam scans a preparation ( laser scanning , English to scan = scan '). Scanning can be done with one point, with several points at the same time or with a line.
The point-by-point rasterization of the specimen can be achieved, for example, by deflecting the laser beam horizontally and vertically by so-called scan mirrors before it is focused on the excitation point in the specimen by the objective . If a three-dimensional image is to be recorded, this is done by creating images of different focal planes one after the other. To do this, either the specimen or the objective is moved in height.
In most cases, generated fluorescence is recorded, so the corresponding devices belong to the fluorescence microscope . The fluorescence-stimulating laser light moves continuously over the specimen, the spatial resolution is created by assigning the fluorescence signal of a certain time segment to an image point. Both the fluorescence intensity and the fluorescence lifetime can be used for image generation , with the latter requiring additional measurement technology ( see also: fluorescence lifetime microscopy , FLIM ). Photomultipliers or avalanche photodiodes are usually used as detectors for scanning with a point , while CCD cameras are used for the other methods . A complete image is never created in the microscope itself. In the case of point detectors this is first put together by the control software, in the case of CCD cameras on the CCD chip.
variants
Different types of laser scanning microscopes are distinguished:
- The historically first laser scanning microscopes were flying spot microscopes , which were replaced by confocal laser scanning microscopes in the 1980s.
- This confocal laser scanning microscopes (Engl. Confocal laser scanning microscope , CLSM) are the most common today. Scanning is mostly done point by point, but there are also variants with several points (spinning disk) or with a line.
- A 4Pi microscope is a variant of the CLSM with improved resolution , which is achieved, among other things, by using two objectives instead of one.
- A STED microscope is also a variant of the CLSM, in which the resolution is improved by the fact that the excitation point is greatly reduced by saturating dye transitions.
- Multiphoton microscopes allow multiphoton fluorescence microscopy and higher harmonic generation .
Depending on the definition of the term “laser scanning microscope”, microscopes can also be included in which strips in the specimen are illuminated in order to record partial images and these strips then change their position. In contrast to the variants described above, however, the movement of the excitation area is not continuous. These include 3D SIM microscopes and light disk microscopy (SPIM or selective plane illumination microscopy ).
See also
literature
- James B. Pawley (Ed.): Handbook of biological confocal microscopy. 3rd edition. Springer Science and Business Media - LLC, New York NY 2006, ISBN 0-387-25921-X .