Spectral self-interference fluorescence microscopy and its applications in biology

An original technique, Spectral Self-Interference Fluorescence Microscopy (SSFM), can determine the location of fluorescent markers above a reflecting surface with sub-nanometer precision. Spontaneous emission of fluorophores located near a mirror is modified by the interference between direct and reflected waves. This leads to an oscillatory pattern in the emission spectrum. The phase and contrast of spectral self-interference oscillations are defined by the position of the emitter with respect to the mirror and the orientation of the corresponding transition dipole. The 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 and orientation of the emitters.; SSFM is able to achieve sub-nanometer sensitivity in measuring the height of a fluorescent monolayer above the reflecting surface. This is demonstrated by determining the surface profile of a silicon oxide chip with a 12nm etched step covered by a monolayer of fluorescein isothiocyanate. In addition, SSFM can be used to resolve the position of a fluorescent marker bound to either the top or the bottom leaflet of a lipid bilayer—a difference in distance is only 4 nm. SSFM can be a valuable tool in studying the conformation of DNA molecules immobilized on surfaces. Determining the location of a fluorescent label at the free end of a tethered to the surface oligonucleotide can clarify the relationship between the spatial orientation of the DNA molecule and the surface charge.; With signal processing of the data, this technique can also be potentially extended to resolving multiple sparse fluorescent layers located as closely as 10 nm. This would push optical microscopy to a new level.