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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]
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 ...
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.
Laser scanning is the controlled deflection of laser beams, visible or invisible. [1] Scanned laser beams are used in some 3-D printers, in rapid prototyping, in machines for material processing, in laser engraving machines, in ophthalmological laser systems for the treatment of presbyopia, in confocal microscopy, in laser printers, in laser shows, in Laser TV, and in barcode scanners.
Fluorescence-lifetime imaging microscopy or FLIM is an imaging technique based on the differences in the exponential decay rate of the photon emission of a fluorophore from a sample. It can be used as an imaging technique in confocal microscopy , two-photon excitation microscopy , and multiphoton tomography.
Photo-activated localization microscopy (PALM or FPALM) [1] [2] and stochastic optical reconstruction microscopy (STORM) [3] are widefield (as opposed to point scanning techniques such as laser scanning confocal microscopy) fluorescence microscopy imaging methods that allow obtaining images with a resolution beyond the diffraction limit.
Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can ...
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]