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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]
Because STED selectively deactivates the fluorescence, it can achieve resolution better than traditional confocal microscopy. Normal fluorescence occurs by exciting an electron from the ground state into an excited electronic state of a different fundamental energy level (S0 goes to S1) which, after relaxing back to the vibrational ground state ...
By virtue of the linearity property of optical non-coherent imaging systems, i.e., . Image(Object 1 + Object 2) = Image(Object 1) + Image(Object 2). the image of an object in a microscope or telescope as a non-coherent imaging system can be computed by expressing the object-plane field as a weighted sum of 2D impulse functions, and then expressing the image plane field as a weighted sum of the ...
This explains why the images for the out-of-focus system (e,f) are more blurry than those of the diffraction-limited system (b,c). Note that although the out-of-focus system has very low contrast at spatial frequencies around 250 cycles/mm, the contrast at spatial frequencies near the diffraction limit of 500 cycles/mm is diffraction-limited.
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]
An automated confocal image reader. High-content screening technology is mainly based on automated digital microscopy and flow cytometry, in combination with IT-systems for the analysis and storage of the data. “High-content” or visual biology technology has two purposes, first to acquire spatially or temporally resolved information on an ...
Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light , and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample.
This formula is suitable for confocal microscopy, but is also used in traditional microscopy. In confocal laser-scanned microscopes, the full-width half-maximum (FWHM) of the point spread function is often used to avoid the difficulty of measuring the Airy disc. [1] This, combined with the rastered illumination pattern, results in better ...