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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]
PSF Lab is a software program that allows the calculation of the illumination point spread function (PSF) of a confocal microscope under various imaging conditions. The calculation of the electric field vectors is based on a rigorous, vectorial model that takes polarization effects in the near-focus region and high numerical aperture microscope objectives into account.
Confocal endoscopy, or confocal laser endomicroscopy (CLE), is a modern imaging technique that allows the examination of real-time microscopic and histological features inside the body. In the word "endomicroscopy", endo- means "within" and -skopein means "to view or observe".
Endomicroscopy is a technique for obtaining histology-like images from inside the human body in real-time, [1] [2] [3] a process known as ‘optical biopsy’. [4] [5] It generally refers to fluorescence confocal microscopy, although multi-photon microscopy and optical coherence tomography have also been adapted for endoscopic use.
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.
The lateral resolution of light sheet fluorescence microscopy can be improved beyond the Abbe limit, by using super resolution microscopy techniques, e.g. with using the fact, that single fluorophores can be located with much higher spatial precision than the nominal resolution of the used optical system (see stochastic localization microscopy ...
[1] [2] A fluorescence microscope is any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image. [3]
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]