Confocal fluorescence detection

Describe, with a labeled diagram, the principle of confocal fluorescent detection. (4 marks)... 

Fig. 3 Schematic representation of a 1000-capillary rotary scanner and a four-color confocal fluorescence detection system. (From Ref. [10].)...

Huang, X. C., Quesada, M. A., and Mathies, R. A. Capillary array electrophoresis using laser-excited confocal fluorescence detection. Anal. Chem. 64 967-972,1992. 

Confocal fluorescence detection allows biological parameters such as the following to be measured quantitatively ... 

Figure 14.3. A survey of read-out modes for confocal fluorescence detection technologies and their applications to biological systems. NA = nucleic acid.

A high-performance confocal fluorescence detection unit usable in either a single channel or multichannel mode. 

Fig. 3 Confocal fluorescence detection on a microchip. A laser was used as the excitation source. The laser light passed through a biconvex lens, was focused into an illumination pinhole, and was subsequently reflected by a dichroic mirror and focused in the channel on the capillary electrophoresis (CE) chip by a microscope objective. The fluorescence signal from the sample, collected by the microscope objective, was passed through the dichroic mirror, focused by the tube lens into a confocal pinhole, and then detected by a photomultiplier tube (PMT). To improve the signal-to-noise ratio, band-pass filter and notch filter were inserted above PMT for spectral filtering...

Dang, F, Zhang, F., Hagiwara, H., Mishina, Y. and Baba, Y. Ultrafast analysis of oligosaccharides on microchip with light-emitting diode confocal fluorescence detection. Electrophoresis, 24, 714, 2003. 

To make CE useful in high-throughput applications, scanning detection and multisheath flow confocal fluorescence detection have been used in commercial 96-capillary array electrophoresis devices. They have greatly facilitated the completion of the Human Genome Project. 

Many researchers using the lectin microarray take advantage of a confocal fluorescence detection principle, because the method has already been adapted for established systems of DNA microarray [18, 19]. The method is simple, and has therefore been widely used. However, it requires washing of the microarray surface after the binding reaction, because a dried sample is required for the confocal-type detection system. One useful application of the system is multicolor detection using, e.g., Cy3 and Cy5, as described later. 

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