Absorption and spontaneous emission of photons by fluorescent molecules are commonly used to probe interactions between biological macromolecules in cells. I will begin this talk with an overview of our development of technologies for determining the supramolecular structure of proteins based on such physical processes. Then, I will outline the physical principles of a proposed method for determining the orientation of single proteins through spectral analysis of spontaneous or stimulated emission.

One of our methods, dubbed Förster Resonance Energy Transfer (FRET) Spectrometry, probes the relative distances between proteins within a complex via quantum energy transfer between the transition dipoles of fluorescent tags attached to them. This is evaluated by detecting the spontaneous emission of photons from both tags. A second method, Fluorescence Intensity Fluctuation (FIF) Spectrometry, probes spatial fluctuations in fluorescence intensities from pixel to pixel in a fluorescence micrograph to determine the number of proteins comprising the complex.

Finally, a new method—still in its infancy—relies on quantifying spectral fluctuations in photon emission by single fluorescent molecules to extract the orientation of their transition dipoles and, through this, the orientation of biomolecules within their natural milieu.

In addition to raising interesting physics-related questions, our studies provide (i) biophysicists and life scientists with tools for understanding cellular signaling and (ii) pharmacologists with in-cell assays for probing the effects of ligands (or drugs) that bind to proteins of interest.