Detection Of Fluorescence And Bioluminescence

Photons derived either from fluorescence or bioluminescence need to be detected and quantified. For imaging purposes, the light derived from labeled proteins contains not only information about the extent of expression, but in the case of fluorescence imaging, also provides where this expression takes place. There are many detectors that can be used to measure light with high accuracy and sensitivity. The following paragraph briefly summarizes the advantages and disadvantages of fluorescence detection methods suitable for studies of GPCR function and trafficking. Photodiodes have the advantage that they exhibit linear responses upon illumination with intensities varying over a large range. In general, they are robust and reliable, and the speed of responses can be quite fast. Avalanche photodiodes for instance are fast enough to measure the lifetime of fluorescence in the femtosecond range. Photodetection by photodiodes is used for a broad spectrum of applications. Photometric detection of FRET or ratiometric detection of Ca2+ by fluorescence indicators are typical examples of their application. Photomultipliers (PMT) have an advantage when detection of light at very low intensities is necessary. Based on their ability to detect light at high frequencies and with excellent quantum yields, PMTs are used in laser scanning microscopes as well as for photometry systems. Charge-coupled devices (CCD) function similarly to an array of photodiodes, and due to their small pixel sizes, they are popular for many imaging devices such as cameras. Technology improvements gave rise to ultrafast and sensitive electron-modifying charge-coupled device (EM-CCD) chips which can take images within a few milliseconds. The sensitivity of these chips has increased over the years, which has helped to increase speed and sensitivity of fluorescence imaging. The application of CCD cameras is very broad and reaches from regular epifluorescence imaging, to single particle tracking, to total internal reflection fluorescence (TIRF) microscopy, to spinning disc confocal microscopy.

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