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A new wide field-of-view confocal imaging system and its applications in drug discovery and pathology

solution wellplate reader. In combination with the capability of imaging large area specimens, automated image acquisition and analysis, the wide field-of-view system can be equipped with multiple slide holders to realize a higher degree of imaging automation. In addition, more efficient methods of image archiving and large image handling and browsing will be developed.

The wide field-of-view confocal imaging system presented in this paper has three excitation lasers and six detection channels, three for fluorescence and reflected-light imaging, and three for bright field. In applications where multiple fluorescence probes and a wider variety of dyes are used, separation of the spectra on the basis of emission wavelengths may not be specific enough, and excitation wavelength must also be selected. Therefore, methods such as the use of an acousto-optic tunable filter (AOTF) for the selection of laser lines may need to be adopted.64 AOTF simultaneously selects the wavelength and controls the degree of attenuation applied.64 When using an AOTF as a tunable dichroic beamsplitter, multiple specific wavelengths can be deflected towards the objectives simultaneously or in sequence. A polarizing beamsplitter can also be used in place of the pri- mary dichroic beamsplitter, which preferentially reflects polarized light (the laser light), while transmitting the nonpolarized fluorescence light to the detection optics.64 When using dyes with overlapping spectra, selection of excitation wavelengths rather than emission wavelengths must also be made. In most cases this can be adjusted sufficiently rapidly to allow "line by line" sequential scanning, where each line is scanned twice - once using one excitation wavelength and one detector, the next using another excitation wavelength and detector.64

Fluorescence detection with dichroic mirrors and barrier filters can be highly specif
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