Genetically encoded fluorescent proteins are an essential tool in cell biology, widely used for investigating cellular processes with molecular specificity. Direct uses of fluorescent proteins include studies of the in vivo cellular localization and dynamics of a protein, as well as measurement of its in vivo concentration. In this chapter, we focus on the use of genetically encoded fluorescent protein as an accurate reporter of in vivo protein numbers. Using the challenge of counting the number of copies of kinetochore proteins in budding yeast as a case study, we discuss the basic considerations in developing a technique for the accurate evaluation of intracellular fluorescence signal. This discussion includes criteria for the selection of a fluorescent protein with optimal characteristics, selection of microscope and image acquisition system components, the design of a fluorescence signal quantification technique, and possible sources of measurement errors. We also include a brief survey of available calibration standards for converting the fluorescence measurements into a number of molecules, since the availability of such a standard usually determines the design of the signal measurement technique as well as the accuracy of final measurements. Finally, we show that, as in the case of budding yeast kinetochore proteins, the in vivo intracellular protein numbers determined from fluorescence measurements can also be employed to elucidate details of cellular structures.
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