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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.
[1] [2] "Fluorescence microscope" refers to 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]
By 1978, the first theoretical ideas had been developed to break the Abbe limit, which called for using a 4Pi microscope as a confocal laser-scanning fluorescence microscope where the light is focused from all sides to a common focus that is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection. [14]
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.
(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.
A 4Pi microscope is a laser scanning fluorescence microscope with an improved axial resolution.With it the typical range of the axial resolution of 500–700 nm can be improved to 100–150 nm, which corresponds to an almost spherical focal spot with 5–7 times less volume than that of standard confocal microscopy.
MINFLUX, or minimal fluorescence photon fluxes microscopy, is a super-resolution light microscopy method that images and tracks objects in two and three dimensions with single-digit nanometer resolution. [1] [2] [3]