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

. The emission filters on the motorized filter wheels can be chosen through software. It is important to have efficient optical filters to block the excitation light from entering the detectors, because the fluorescence light is about 1,000 times weaker than the excitation. The three detection channels can be used in simultaneous or sequential acquisition modes. For example, simultaneous fluorescence detection can be used for colocalization analysis; sequential detection can be used to decouple fluorescence spectra when multiple fluorophores are present. Although more time consuming, sequential detection has the advantage of reducing bleed-through of the emission spectrum of one fluorochrome into that of another (cross talk), which results in false positive imaging.

In fluorescence microscopy of thin specimens, although there is little interference from neighboring focal planes within the specimen, a confocal system does provide higher sensitivity and offers sharp images because of its ability to reject glare from the slide substrate and stray light from other sources.

2.4. Reflection Imaging

Confocal microscopy is mostly used for fluorescence imaging in biomedical research, in consideration of its optical sectioning power combined with the specificity of multiple fluorescence labeling to produce improved images of cells and tissues. However, the confocal imaging system can also be used in reflectance microscopy to gather additional information from a specimen. Confocal reflectance microscopy provides information on unstained tissues and those labeled with probes that reflect light. For example, unlabeled cells can be detected in a population of fluorescently labeled cells, or the interactions between fluorescently labeled cells growing on an opaque and patterned substrate can be imaged.55

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