Imaging of Molecular Complexes on the Surfaces of Living Cells by Uniform Total Internal Reflection-Based Fret Microscopy

Abstract

AbstractFluorescence Resonance Energy Transfer (FRET) microscopy measures the interaction between donor and acceptor labeled proteins in living cells. Currently, these measurements are limited by out-of-focus contributions, which degrade the image. Total internal reflection (TIR) microscopy can selectively excite molecules within about 150 nanometers of the glass-water interface thereby eliminating out-of-focus fluorescence. However, the interference fringing of the coherent laser illumination used in TIR creates artifacts that prohibit quantitative imaging methods that require multiple laser illuminations such as FRET. Axelrod and collogues demonstrated that for through-the-lens TIR that rapid azimuthal spinning of a collimated beam in the back focal plane eliminates interference fringes. Here we describe multicolor, depth-matched, 360-degree spinning TIR illumination to enable quantitative, high-resolution FRET imaging of molecular complexes near the plasma membranes of living cells. We have devised new methods for normalizing spatial variations in illumination and calibrating this field for quantitative FRET analysis of protein interactions on the plasma membrane. Initial applications to Rac1 activation demonstrate a novel spatial organization of these signaling activities during adhesion of macrophages to the coverglass.