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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]
The signal can be acquired with a camera in wide-field operation (a, b) or by point detection in confocal arrangement (c, d). Interferometric scattering microscopy ( iSCAT ) refers to a class of methods that detect and image a subwavelength object by interfering the light scattered by it with a reference light field.
With no modification to the microscope, i.e. with a simple wide field light microscope, the quality of optical sectioning is governed by the same physics as the depth of field effect in photography. For a high numerical aperture lens, equivalent to a wide aperture, the depth of field is small (shallow focus) and gives good optical sectioning.
[1] [2] "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image. [3]
Conventional, widefield microscopy is generally unsuitable for imaging thick tissue because the images are corrupted by a blurred, out-of-focus background signal. [10] Endomicroscopes achieve optical sectioning (removal of the background intensity) using the confocal principle - each image frame is assembled in a point-by-point fashion by ...
Widefield fluorescence was introduced in 1910 which was an optical technique that illuminates the entire sample. [3] 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.
The three-dimensional point spread functions (a,c) and corresponding modulation transfer functions (b,d) of a wide-field microscope (a,b) and confocal microscope (c,d). In both cases the numerical aperture of the objective is 1.49 and the refractive index of the medium 1.52.
Photo-activated localization microscopy (PALM or FPALM) [1] [2] and stochastic optical reconstruction microscopy (STORM) [3] are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit.