Confocal scanning-beam laser

Figure 14.4. Confocal scanning-beam laser microscope. (From Ref. 6, with permission from the Electrochemical Society.)...

Figure 14.4 gives the optical system particulars of the CSBLM (confocal scanning-beam laser microscope) used. The reader should keep in mind that a detailed understanding of this system requires a good knowledge of optics. Some readers may choose to skip the next section on first reading. 

Commercial confocal and two-photon laser scanning microscopes scan the laser beam by fast galvano-driven mirrors. Typical pixel dwell times are of the order of a few microseconds. Depending on the number of pixels, a complete frame is scanned within 25 ms to several seconds. 

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... 

Fig. 6.1 Schematic of a laser confocal scanning microscope. The laser illumination spot is scanned across the sample. Reflected light is de-scanned and passed through the beam splitter and the confocal aperture to the detector. The detector of transmitted light may be present as an accessory, but it gives a normal, not a confocal, optical microscope image.

Type 3, scanning beam A focused beam (laser tight or electron beam) scans across the specimen, resulting in a reflected beam from the surface (as in a confocal laser scanning microscope) or in secondary or backscattered electrons (in scanning electron microscopes) thick and thin specimens can be studied. 

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. 

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