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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]
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.
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.
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 ...
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century.
Diagram illustrating near-field optics, with the diffraction of light coming from NSOM fiber probe, showing wavelength of light and the near-field. [1] Comparison of photoluminescence maps recorded from a molybdenum disulfide flake using NSOM with a campanile probe (top) and conventional confocal microscopy (bottom). Scale bars: 1 μm. [2]
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The ability of a lens to resolve detail is usually determined by the quality of the lens, but is ultimately limited by diffraction.Light coming from a point source in the object diffracts through the lens aperture such that it forms a diffraction pattern in the image, which has a central spot and surrounding bright rings, separated by dark nulls; this pattern is known as an Airy pattern, and ...