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Scanning acoustic microscopy works by directing focused sound from a transducer at a small point on a target object. Sound hitting the object is either scattered, absorbed, reflected (scattered at 180°) or transmitted (scattered at 0°). It is possible to detect the scattered pulses travelling in a particular direction.
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 notion of acoustic microscopy dates back to 1936 when S. Ya. Sokolov [1] proposed a device for producing magnified views of structure with 3-GHz sound waves. However, due to technological limitations at the time, no such instrument could be constructed, and it was not until 1959 that Dunn and Fry [2] performed the first acoustic microscopy experiments, though not at very high frequencies.
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.
[1] [2] A fluorescence microscope is 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]
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution. With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy .
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