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Chapter 8:
Review of Available Instrumentation for In Vivo Bioluminescence and Fluorescence Imaging

Two main groups of instruments suitable for in vivo fluorescence and bioluminescence imaging are available on the market: plate readers and imaging devices. The main difference between the two is that plate readers provide an integrated light signal from the well-which is detected by photodiodes or photomultiplier tubes (PMTs)-while imaging devices use CCD cameras as light detectors and are able to register not only intensity but spatial distribution in time. Imaging devices are made for both microscopic and macroscopic formats, with the latter capable of imaging the whole body of a small animal. The major features and limitations of these types of instrumentation are presented below.

Raw data in all types of devices designed for measurement of light emission (fluorescence and bioluminescence) are presented in relative light units (RLUs). RLU is not a scientifically defined unit but rather a measure of relative light output from a sample. The software in some imaging devices provides the means to convert RLU values to absolute photon counts. This feature should be treated with great caution. In general, the conversion coefficient depends on the wavelength of light, the geometry and relative positions of the object and the detector, and to a lesser extent, on the optical properties of materials used in construction of the measuring chamber. Taking into consideration all variables in each experimental setup, it is clear that for the conversion coefficient to be accurate, it should be established for each particular case. One of the ways to compare readings for the same sample obtained from different equipment in each instrument is to calibrate the instruments individually, using a characterized light source with the geometry and location similar to those of the sample.

Despite this uncertainty in defining absolute values of measured light intensity, use of relative units allows very accurate measurements of even small changes in light output. Including appropriate controls in an experimental setup permits accurate quantification of the obtained data when other optical conditions remain constant.

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