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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 .
Scanning laser ophthalmoscopy developed as a method to view a distinct layer of the living eye at the microscopic level. The use of confocal methods to diminish extra light by focusing detected light through a small pinhole made possible the imaging of individual layers of the retina with greater distinction than ever before. [4]
Additional flexibility can be added by using digital light-sheet microscopy to generate the illumination patterns. In digital LSM, the light sheet is created by rapidly scanning a laser beam through the sample. [3] [2] [14] This allows for fine control over the specific illumination pattern by modulating the intensity of the laser as it scans ...
Recent developments like scanning laser ophthalmoscopy can make good quality images through pupils as small as 2 mm (0.079 in), so dilating the pupil is not necessary with these methods. [ citation needed ]
Three-photon microscopy (3PEF) is a high-resolution fluorescence microscopy based on nonlinear excitation effect. [1] [2] [3] Different from two-photon excitation microscopy, it uses three exciting photons. It typically uses 1300 nm or longer wavelength lasers to excite the fluorescent dyes with three simultaneously absorbed photons.
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.
Scanning probe microscopy (SPM) is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. SPM was founded in 1981, with the invention of the scanning tunneling microscope , an instrument for imaging surfaces at the atomic level.
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