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Laser microscopy (or laser scanning microscopy) is a class of techniques for generating microscopic images of some sample by raster scanning it with a diffraction-limited laser beam. Scanning may be achieved by moving either the laser beam or the sample. Typically, the laser beam excites fluorescence in its focus, and the intensity of that fluorescence light is recorded for each point in the sample (→ fluorescence microscopy). From these data, images can be produced on a computer, and of course they can be stored in electronic form. Numerical methods can be applied to process the images, e.g. to enhance the contrast.
A frequently used imaging technique is based on a confocal geometry , where the light from the focus in the sample is imaged (e.g. with a microscope objective) onto a pinhole, behind which the optical power is detected. This geometry suppresses the influence of light coming from other regions in the sample, e.g. from before or after the focus, because such light can not efficiently pass through the pinhole. In effect, mainly the depth resolution is improved. The confocal principle is emphasized in the term confocal laser scanning microscopy, and discussed in more detail in the article on fluorescence microscopy.
Instead of fluorescence, one may exploit acoustic effects of pulsed laser beams; the resulting method is called optoacoustic or photoacoustic microscopy .
Methods for Sub-diffraction Resolution
For most variants of laser microscopy, diffraction limits the possible beam waist radius of the scanning laser beam and thus the obtained image resolution. However, some techniques allow to beat the diffraction limit, and are sometimes called super-resolution microscopy or nanoscopy. One class of methods is based on non-uniform illumination in combination with a nonlinear photoresponse. Stimulated emission depletion microscopy (STED microscopy) (explained in the article on fluorescence microscopy, and related to the Nobel Prize in Chemistry 2014) is the most popular method of that type. Other techniques are based on certain fluorescent molecules, which preferably occupy certain parts of the specimen and can be localized very precisely (→ stochastic optical reconstruction microscopy, STORM).
|||W. Denk et al., “Two-photon laser scanning fluorescence microscopy”, Science 248, 73 (1990)|
|||R. H. Webb, “Confocal optical microscopy”, Rep. Prog. Phys. 59 (3), 427 (1996)|
|||J. Squier et al., “Third harmonic generation microscopy”, Opt. Express 3 (9), 315 (1998)|
|||L. V. Wang, “Multiscale photoacoustic microscopy and computed tomography”, Nature Photon. 3 (9), 503 (2009)|
|||Nature Photon. 3 (7), special issue on super-resolution imaging (2009)|
|||National High Magnetic Field Laboratory, “Optical microscopy primer on fluorescence microscopy”, http://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html|
|||K. W. Dunn et al., “Fundamental concepts in confocal microscopy”, http://www.microscopyu.com/articles/confocal/index.html|
|||S. W. Hell, “Nobel Lecture: Nanoscopy with freely propagating light”, Rev. Mod. Phys. 87, 1169 (2015)|
See also: fluorescence microscopy
and other articles in the category methods
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