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Confocal microscopy

Research and commercial application of confocal microscopy has witnessed tremendous development in recent years. In the confocal arrangement, the sample of interest (a biological tissue specimen or a spot on a micro-array) may be illuminated with white light or laser wavelengths relayed through a high NA imaging objective to realize a small focal volume. A fluorescent marker within the illuminated sample volume gives rise to the signal of interest. In the light collection optical path exists a pinhole, or confocal, aperature. This arrangement allows for isolation of fluorescence within the image plane and also reduction of background signal. The enhancement of signal to noise made possible by the confocal aperature allows for detailed 3D scanning of complex biological specimens or fast scanning of high-density microarrays.

In terms of light detection, a detector with excellent quantum efficiency and fast response is necessary because of the small illumination volume, weak intensity of the fluorescence, and high scanning speed. Wavelengths of 400 to 500 nm are typically of interest, although response at longer wavelengths may also be important with methods such as FRET. Photomultiplier tubes are ideally suited to confocal microscopy demands, and APD modules and CCD cameras are also commonly used. The ideal detector choice depends on the sample and imaging requirements of the application.

 

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