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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]
Köhler illumination is a method of specimen illumination used for transmitted and reflected light (trans- and epi-illuminated) optical microscopy.Köhler illumination acts to generate an even illumination of the sample and ensures that an image of the illumination source (for example a halogen lamp filament) is not visible in the resulting image.
Additional flexibility can be added by using digital light-sheet microscopy to generate the illumination patterns. In digital LSM, the light sheet is created by rapidly scanning a laser beam through the sample. [3] [2] [14] This allows for fine control over the specific illumination pattern by modulating the intensity of the laser as it scans ...
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide.
Also the good optical sectioning capability reduces the background signal and thus creates images with higher contrast, comparable to confocal microscopy. Because light sheet fluorescence microscopy scans samples by using a plane of light instead of a point (as in confocal microscopy), it can acquire images at speeds 100 to 1,000 times faster ...
A simple microscope uses the optical power of a single lens or group of lenses for magnification. A compound microscope uses a system of lenses (one set enlarging the image produced by another) to achieve a much higher magnification of an object. The vast majority of modern research microscopes are compound microscopes, while some cheaper ...
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique based on the differences in the exponential decay rate of the photon emission of a fluorophore from a sample. It can be used as an imaging technique in confocal microscopy , two-photon excitation microscopy , and multiphoton tomography.
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). Although the same microscope objective with a numerical aperture of 1.49 is used, it is clear that the confocal point spread function is more compact both in the lateral dimensions ...