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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 4Pi microscope is a laser-scanning fluorescence microscope with an improved axial resolution. The typical value of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy .
Here, λ 0 is the wavelength in vacuum; NA is the numerical aperture for the optical component (maximum 1.3–1.4 for modern objectives with a very high magnification factor). Thus, the resolution limit is usually around λ 0 /2 for conventional optical microscopy. [17]
An account of the early history of scanning electron microscopy has been presented by McMullan. [2] [3] Although Max Knoll produced a photo with a 50 mm object-field-width showing channeling contrast by the use of an electron beam scanner, [4] it was Manfred von Ardenne who in 1937 invented [5] a microscope with high resolution by scanning a very small raster with a demagnified and finely ...
Two-photon excitation microscopy of mouse intestine.Red: actin.Green: cell nuclei.Blue: mucus of goblet cells.Obtained at 780 nm using a Ti-sapphire laser.. Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that is particularly well-suited to image scattering living tissue of up to about one millimeter in thickness.
One of the most important properties of microscope objectives is their magnification.The magnification typically ranges from 4× to 100×. It is combined with the magnification of the eyepiece to determine the overall magnification of the microscope; a 4× objective with a 10× eyepiece produces an image that is 40 times the size of the object.
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