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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 ...
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]
Gaussian beam width () as a function of the axial distance .: beam waist; : confocal parameter; : Rayleigh length; : total angular spread In optics and especially laser science, the Rayleigh length or Rayleigh range, , is the distance along the propagation direction of a beam from the waist to the place where the area of the cross section is doubled. [1]
Structured illumination microscopy (SIM) is a method of super-resolution microscopy which is performed by acquiring multiple images of the same sample under different patterns of illumination, then computationally combining these images to achieve a single reconstruction with up to 2x improvement over the diffraction limited lateral resolution.
In fluorescence microscopy the excitation and emission are typically on different wavelengths. In total internal reflection fluorescence microscopy a thin portion of the sample located immediately on the cover glass is excited with an evanescent field, and recorded with a conventional diffraction-limited objective, improving the axial resolution.