Confocal scanning laser microscopy principle

S. Wilhelm, B. Grdbler, M. Gluch, H. Heinz. Confocal laser scanning microscopy principles, www.zeiss.de/lsm, 2003. 

In the present chapter, we discuss the principles and techniques commonly used for observing biological surface structures, including optical microscopy (light microscopy, laser scanning confocal microscopy), electron microscopy (scanning electron microscopy, transmission electron microscopy), and scanning probe microscopy. We describe and contrast the sample preparation of each technique. Quantitative data analysis as well as the limitations of each technique is also addressed. 

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...

Fig.1 Schematic representing the optical pathway and image information flow of the laser scanning confocal microscopy (LSCM) technique. All principle components of generic LSCM are labeled (Redrawn in part frtnn Carl Zeiss The Confocal Laser Scanning Microscope)...

Multiphoton or two-photon laser scanning microscopy is an alternative to confocal and time-resolved microscopy for bioimaging applications. The principle has been discussed in Lanthanides Luminescence Applications and concerns a two-photon excitation from the simultaneous absorption of two photons in a single quantized event. A bioprobe that normally absorbs ultraviolet light (Xex = 350 nm) can also be excited by two photons of NIR light, at 700 nm (the wavelength is twice that required for one-photon excitation). These two photons must interact simultaneously, which means in a very small lapse time. The instrumentation requires pulse lasers to provide sufficient power, as the photon density must... 

Principles and Characteristics Diffraction limits the spatial resolution of conventional optical microscopy instruments. In practice, the resolution limit is approximately 0.6A., i.e. about 0.5 pm for optical microscopes. Resolution in far-field optical microscopy techniques may be improved (though slightly) by the application of UV (cfr. Chp. 5.3.2) or confocal laser scanning (c/r Chp. 5.3.4). For confocal laser imaging with green light (A. = 500 nm), resolution is limited... 

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