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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]
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]
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.
Confocal laser scanning microscopy uses a focused laser beam (e.g. 488 nm) that is scanned across the sample to excite fluorescence in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a photomultiplier tube. The image is constructed in a computer ...
Edge filters are used for removal of all primary laser light. Resolution as low as 10 nm can be achieved using this technique. [citation needed] Near field infrared spectrometry and near-field dielectric microscopy [19] use near-field probes to combine sub-micron microscopy with localized IR spectroscopy. [25]
Medical optical imaging is the use of light as an investigational imaging technique for medical applications, pioneered by American Physical Chemist Britton Chance.Examples include optical microscopy, spectroscopy, endoscopy, scanning laser ophthalmoscopy, laser Doppler imaging, optical coherence tomography, and transdermal optical imaging.
Confocal microscope, a widely used variant of epifluorescent illumination that uses a scanning laser to illuminate a sample for fluorescence. Two-photon microscope, used to image fluorescence deeper in scattering media and reduce photobleaching, especially in living samples.
Non-invasive phase-contrast techniques are therefore often used as a vital complement to fluorescent microscopy in live-cell imaging applications. [16] [17] Deep learning-assisted fluorescence microscopy methods, however, help to reduced light burden and phototoxicity and allow even repeated high resolution live imaging. [18]
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