Spectral Self-Interference Fluorescence Microscopy for Subcellular Imaging

Spectral self-interference fluorescence microscopy (SSFM) has been recently developed to determine the axial position of fluorescent emitters placed on reflecting dielectric structures. In this paper, we review SSFM with emphasis on its axial localization capabilities. We show that there is a tradeoff between the numerical aperture (NA) of the microscopy system and the axial localization fidelity. To use high-NA objectives for better lateral resolution, we describe and demonstrate a high-NA 4Pi microscopy system that performs spectral self-interference microscopy for axial localization of less than 2 nm. We demonstrate axial localization by using artificial samples. We also probe the membrane topography of a Shigella flexneri bacterium, several micrometers away from a solid support.