FRET by fluorescence polarization microscopy.

The widespread success in using genetically encoded fluorescent proteins (FPs) to track protein motion in living cells has led to extensive interest in measuring Förster resonance energy transfer (FRET) between two FPs of different colors. FRET occurs over distances less than 10-nm and can thus be used to detect protein-protein interactions and changes in protein conformation. However, FP-FRET measurements are complicated by the spectral properties of FPs. Consequently, extensive correction or photo-destructive approaches have been used to detect the presence of FRET. Since these methods limit the temporal and spatial resolution of FRET measurements, they are not well suited for many live-cell imaging applications. Here, we describe an alternative approach to detect FP-FRET by measuring fluorescence anisotropies (AFRET). Since FPs are large in size, excitation of FPs with polarized light results in highly polarized emission. In this case, FRET to a second FP that lies outside the photoselection plane will depolarize the fluorescence. This method provides high contrast and unambiguous indication of FRET using a simple image collection strategy that can be easily adapted to any modality including widefield and laser scanning approaches. In this chapter, we will discuss the theory behind AFRET imaging, calculation of FP anisotropies using fluorescent microscopes, and configuration of microscopes for AFRET experiments.