Many components of confocal microscopes have been described in the previous chapters. In this chapter I will briefly consolidate the key points and provide some additional critical details.
The selection of components should be optimized for the type of specimen to be observed and for the imaging mode, i.e., reflected light or fluorescence. The clinical applications of confocal microscopy to ophthalmology and dermatology are predominantly based on reflected light. However, the biological applications of confocal microscopy are chiefly based on fluorescence techniques. These applications include imaging specific proteins based on green fluorescent protein (GFP) fluorescence, fluorescent indicator dyes that respond to ion concentration and transmembrane voltage differences, fluorescent labeled antibodies to image specific proteins, and fluorescent membrane dyes.
As new fluorescent probes are developed, there may be a need to change the light source to match their absorption bands. Similarly, new dichroic mirrors and filters may be required. New types of detectors are facilitating new techniques; multi-grid photomultipliers in conjunction with fluorescence dispersion devices, and the quantum efficiency and SNR of two-dimensional detectors is improving along with their spectral range. Manufacturers are responding to their users by developing microscope objectives that are optimized for specific applications of confocal microscopy, e.g., neurobiology and developmental biology.
9.1 Light Sources
Light is defined as the visible form of electromagnetic radiation. In the light microscope it is important to make use of these specific characteristics of electromagnetic radiation: wavelength, polarization, and coherence. When choosing a light source one must evaluate broadband or white light sources versus narrowband sources; noise and stability; and the cost of cooling, replacement, and maintenance.
Again, the first question to ask is: What type or types of specimen will be imaged? Is the microscope to be dedicated to imaging integrated circuits? Is it to be used in the clinic to image in vivo human skin or the living human eye? What types of fluorescent probes are used with the specimen? What are the absorption bands of the fluorescent probes that will be studied?
White light sources are necessary for real-color direct-view Nipkow disk-based confocal microscopes.
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