Design and validation of a new confocal FRET polarization anisotropy microscope

INTRODUCTION Protein interactions constitute a fundamental process in almost all regulatory pathways in living cells but are highly challenging with regards to experimental accessibility. With the use of GFP and the production of numerous kinds of fluorescent derivates it became possible to measure Förster Resonance Energy Transfer (FRET) between fluorescently labelled proteins in living cells. FRET can be detected via different techniques. Most popular are sensitized emission, acceptor photobleaching and fluorescence lifetime imaging. Here we present a setup to measure FRET by detecting fluorescence polarization anisotropy. The system is based on a Yokogawa spinning disk confocal microscope. The setup is shown and validated with live cell measurements of GFP and mCherry constructs.