Confocal scanning-beam laser microscopy

Confocal scanning laser microscopy has been combined with the use of fluorescent stains to measure the distribution and concentration of specific analytes within biological tissue. Stains are used to bind selectively with the analyte under study, and the correct laser beam is selected to excite the stain used to give a fluorescent signal. The technique can also be... 

Type 3 - Scanning beam A focused beam (laser light or electron beam) is seanned aeross the speeimen, resulting in a refleeted beam from the surfaee (as in confocal laser scanning microscopy) or in secondary or backscattered electrons (BSE in scanning electron microscopes). 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Scanning the surface with a laser beam in a confocal arrangement can result in topographic information about the investigated surface since the confocal arrangement and further optical features dominate this experimental approach, whereas the scanning feature is of lesser importance, this microscopy is treated in the Sect. 7.3. 

Figure 11.9 Schematic illustration of the principles of confocal fluorescence microscopy singlephoton exdted fluorescence occurs along the entire beam path, however the insertion of a pinhole in the focal plane of the objective ensures that fluorescence from outside the focal point of the laser (dashed line) is prevented from reaching the detector. The laser is scanned in theX-K plane to build up an image of the sample for a given depth. Translation of the sample in the Z-direction allows images (optical sections) to be recorded as a function of depth...

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