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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]
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 ...
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Raman microscopy, and in particular confocal microscopy, can reach down to sub-micrometer lateral spatial resolution. [7] Because a Raman microscope is a diffraction-limited system, its spatial resolution depends on the wavelength of light and the numerical aperture of the focusing element. In confocal Raman microscopy, the diameter of the ...
An example of an experimentally derived point spread function from a confocal microscope using a 63x 1.4NA oil objective. It was generated using Huygens Professional deconvolution software. Shown are views in xz, xy, yz and a 3D representation. In microscopy, experimental determination of PSF requires sub-resolution (point-like) radiating sources.
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.
(a) Optically sectioned fluorescence images of a pollen grain. (b) Combined image. (c) Combined image of a group of pollen grains. [1]Optical sectioning is the process by which a suitably designed microscope can produce clear images of focal planes deep within a thick sample.
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.