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Fluorescence and confocal microscopes operating principle. Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. [1]
(a) Optically sectioned fluorescence images of a pollen grain. (b) Combined image. (c) Combined image of a group of pollen grains. [1]Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample.
Widefield fluorescence was introduced in 1910 which was an optical technique that illuminates the entire sample. [3] Confocal microscopy was then introduced in 1960 which decreased the background and exposure time of the sample by directing light to a pinpoint and illuminating cones of light into the sample.
Photo-activated localization microscopy (PALM or FPALM) [1] [2] and stochastic optical reconstruction microscopy (STORM) [3] are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit.
The image of a point source is also a three dimensional (3D) intensity distribution which can be represented by a 3D point-spread function. As an example, the figure on the right shows the 3D point-spread function in object space of a wide-field microscope (a) alongside that of a confocal microscope (c).
Several types of microscope are regularly used: widefield, confocal, or two-photon. Most microscopy system will also support the collection of time-series (movies). In general, filters are used so that each dye is imaged separately (for example, a blue filter is used to image Hoechst, then rapidly switched to a green filter to image GFP).
The signal can be acquired with a camera in wide-field operation (a, b) or by point detection in confocal arrangement (c, d). Interferometric scattering microscopy ( iSCAT ) refers to a class of methods that detect and image a subwavelength object by interfering the light scattered by it with a reference light field.
Antonie van Leeuwenhoek (1632–1723). The field of microscopy (optical microscopy) dates back to at least the 17th-century.Earlier microscopes, single lens magnifying glasses with limited magnification, date at least as far back as the wide spread use of lenses in eyeglasses in the 13th century [2] but more advanced compound microscopes first appeared in Europe around 1620 [3] [4] The ...