Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with L_p-regularized image reconstruction

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (10²-10⁶ W/cm²), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80nm with illumination power densities as low as ~1W/cm² for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L₁-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the L_p-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of superresolution fluorescence microscopy for live-cell imaging.