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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]
Stimulated emission depletion (STED) microscopy is one of the techniques that make up super-resolution microscopy. It creates super-resolution images by the selective deactivation of fluorophores , minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. [ 1 ]
Scanning confocal electron microscopy (SCEM) is an electron microscopy technique analogous to scanning confocal optical microscopy (SCOM). In this technique, the studied sample is illuminated by a focussed electron beam, as in other scanning microscopy techniques, such as scanning transmission electron microscopy or scanning electron microscopy .
The wave nature of light limits the size of the spot to which light can be focused due to the diffraction limit. This limitation was described in the 19th century by Ernst Abbe and "limits an optical microscope's resolution to approximately half of the wavelength of the light used." Fluorescence microscopy is central to many techniques which ...
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 ...
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. In the 1980s, the introduction of TIRFM further decreased background and exposure time by only illuminating the thin section of the sample being examined ...
In these techniques light is focused on a sample and the measured fluorescence intensity fluctuations (due to diffusion, physical or chemical reactions, aggregation, etc.) are analyzed using the temporal autocorrelation. Because the measured property is essentially related to the magnitude and/or the amount of fluctuations, there is an optimum ...
It is usually done in the digital domain by a software algorithm, as part of a suite of microscope image processing techniques. Deconvolution is also practical to sharpen images that suffer from fast motion or jiggles during capturing. Early Hubble Space Telescope images were distorted by a flawed mirror and were sharpened by deconvolution.