Instrumentation confocal laser scanning

The classical polarizing light microscope as developed 150 years ago is still the most versatile, least expensive analytical instrument in the hands of an experienced microscopist. Its limitations in terms of resolving power, depth of field, and contrast have been reduced in the last decade, in which we have witnessed a revolution in its evolution. Video microscopy has increased contrast electronically, and thereby revealed structures never before seen. With computer enhancement, unheard of resolutions are possible. There are daily developments in the X-ray, holographic, acoustic, confocal laser scanning, and scanning tunneling micro-... 

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... 

Confocal laser scanning microscopy (CLSM) has been applied mainly in biology [58], For example, introducing a fluorescence probe in a cell, a specific portion of the cell is stereoscopically observed. Today, this instrument is used not only in biology but also has begun to see widespread use in the materials research field [59]. 

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... 

A wide range of techniques can be used to capture the surface roughness of a component using noncontact methods. Some of the more common instruments used to captore topographic data include confocal microscopy, laser triangulation, focus detection, and optical interferometry. A relatively recent branch of microscopy known as scanning probe microscopy (SPM) yields over 20 other instruments which are defined based on what probe-surface interaction they are monitoring. The SPM family is described in more detail elsewhere in this book and will be briefly mentioned in this article. 

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