High resolution spectral self-interference fluorescence microscopy

We present a new method of fluorescence imaging, which yields nm-scale axial height determination and ~15 nm axial resolution. The method uses the unique spectral signature of the fluorescent emission intensity well above a reflecting surface to determine vertical position unambiguously. We have demonstrated axial height determination with nm sensitivity by resolving the height difference of fluorescein directly on the surface or ontop of streptavidin. While different positions of fluorophores of different color are determined independently with nm precision, resolving the position of two fluorophores of the same color is a more convoluted problem due to the finite spectral emission widow of the fluorophores. Hence, for physically close (<λ/2) fluorophores, it is necessary to collect multiple spectra by independently scanning an excitation standing wave in order to deconvolute the contribution to the spectral pattern from different heights. Moving the excitation standing wave successively enhances or suppresses excitation from different parts of the height distribution, changing the spectral content. This way two fluorophores of the same color can be resolved to better than 20 nm. Design aspects of the dielectric stack for independent excitation wave scanning and limits of deconvolution for an arbitrary height distribution will be discussed.