FRET (Förster Resonance Energy Transfer) microscopy breaks the resolution limit of light to let us investigate the conformation and function of proteins within living cells. Intensity-based methods are the most popular and direct approach to detect FRET. Among them, detection of sensitized emission signals and ratio imaging of specially designed FRET sensors are routinely used in modern cell biology laboratories. In this chapter, we provide protocols for both these techniques. We guide the reader through the mathematical corrections necessary to calculate the sensitized emission image. We illustrate this approach with an example of studying the interaction of nexin (SNX1) proteins. In the ratio FRET protocol, we focus on monitoring changes in cellular concentration of cAMP with an EPAC-based FRET sensor.
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