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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.
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]
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.
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.
The image of a point source is also a three dimensional (3D) intensity distribution which can be represented by a 3D point-spread function. As an example, the figure on the right shows the 3D point-spread function in object space of a wide-field microscope (a) alongside that of a confocal microscope (c).
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.
Several types of microscope are regularly used: widefield, confocal, or two-photon. Most microscopy system will also support the collection of time-series (movies). In general, filters are used so that each dye is imaged separately (for example, a blue filter is used to image Hoechst, then rapidly switched to a green filter to image GFP).
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 ...