Probing bacterial surfaces using 4Pi spectral self-interference fluorescence microscopy

We present nanometer scale axial localization of fluorescent markers to probe subcellular structures using self-interference based fluorescence microscopy. We show probing the membrane topography of a gram-negative bacterium, Shigella flexneri, and discuss further applications.

Along with the advances in the fluorescence imaging techniques [1]–[3], recently developed fluorescence localization techniques [4]–[6] opened a new avenue for the high resolution optical study of subcellular structures. While these techniques localize emitters in the lateral dimensions, axial localization of fluorescence has also been shown on reflective substrates by exploiting the interference of excitation and the self-interference of fluorescence emission. Among these techniques, fluorescence interference contrast (FLIC) microscopy [7] utilizes the changes in the modified total emission intensity as a function of position whereas spectral self-interference fluorescence microscopy (SSFM) [8] relies on the spectral oscillations in the emission spectrum to identify the axial position of emitters. Unlike in FLIC microscopy, axial localization in SSFM is insensitive to bleaching or non uniform labeling that may be misinterpreted as axial position change. However, in order to have sufficient contrast in the spectral oscillations to determine axial position with nanometer precision, SSFM systems require low NA objectives and their lateral resolution is poor.