Abstract
We present a method and an apparatus of polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging microscopy done in parallel for improved interpretation of the photophysical interactions. We demonstrate this apparatus to better determine the protein–protein interactions in the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G‐protein coupled receptor, both fused to the cyan and yellow fluorescent proteins for either inter‐ or intramolecular FRET. In both cases, the expression levels of proteins and also background autofluorescence played a significant role in the depolarization values measured in association with FRET. The system has the sensitivity and low‐noise capability of single‐fluorophore detection. Using counting procedures from single‐molecule methods, control experiments were performed to determine number densities of green fluorescence protein variants CFP and YFP where homo resonance energy transfer can occur. Depolarization values were also determined for flavins, a common molecule of cellular background autofluorescence. From the anisotropy measurements of donor and acceptor, the latter when directly excited or when excited by energy transfer, we find that our instrumentation and method also characterizes crucial effects from homotransfer, polarization specific photobleaching and background molecules. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.