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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]
This technique, called TRAFIX, enabled wide-field imaging through biological tissue at great depths with higher signal-to-background ratio and lower photobleaching than standard point-scanning two-photon microscopy. [13] Another simpler method consists of two beams that are loosely focused and overlapped onto an area (~100 μm) on the sample.
The three-dimensional point spread functions (a,c) and corresponding modulation transfer functions (b,d) of a wide-field microscope (a,b) and confocal microscope (c,d). In both cases the numerical aperture of the objective is 1.49 and the refractive index of the medium 1.52.
A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide.
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 ...
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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.
Intravital microscopy can be performed using several light microscopy techniques including widefield fluorescence, confocal, multiphoton, spinning disc microscopy and others. The main consideration for the choice of a particular technique is the penetration depth needed to image the area and the amount of cell-cell interaction details required.