Biological applications of spectral self-interference

An original technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), can determine the location of fluorescent markers above a reflecting surface with sub-nanometer precision. SSFM was used to resolve the position of a fluorescent marker bound to either the top or the bottom leaflet of a lipid bilayer – the difference in distance is only 4 nm. SSFM is a valuable tool in studying the conformation of DNA molecules immobilized on surfaces. A fluorescent label attached to a DNA molecule tethered to the surface can help elucidate its spatial orientation. This method is based on the fact that spontaneous emission of fluorophores located near a mirror is modified by the interference between direct and reflected waves, which leads to an oscillatory pattern in the emission spectrum. Spectral patterns of emission near surfaces can be precisely described with a classical model that considers the relative intensity and polarization state of direct and reflected waves depending on dipole orientation. An algorithm based on the emission model and polynomial fitting built into a software application can be used for fast and efficient analysis of self-interference spectra yielding information about the location of the emitters with very high precision.