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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.
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 ...
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]
The near-field optical (NFO) microscope involved a sub-wavelength aperture at the apex of a metal coated sharply pointed transparent tip, and a feedback mechanism to maintain a constant distance of a few nanometers between the sample and the probe. Lewis et al. were also aware of the potential of an NFO microscope at this time. [14]
The principle setup of a light sheet fluorescence microscope. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique with an intermediate-to-high [1] optical resolution, but good optical sectioning capabilities and high speed.
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In microscopy, NA generally refers to object-space numerical aperture unless otherwise noted. In microscopy, NA is important because it indicates the resolving power of a lens. The size of the finest detail that can be resolved (the resolution) is proportional to λ / 2NA , where λ is the wavelength of the light. A lens with a larger ...