Highly-Biocompatible Multiplexed Super-Resolution Imaging by SPoD-OnSPAN

Although super-resolution microscopy enables optical imaging beyond the diffraction limit of light, achieving super-resolution imaging with high biocompatibility remains a significant challenge. Conventional super-resolution techniques, such as stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOLFT), and single-molecule localization microscopy (SMLM), require relatively high illumination power densities, typically ranging from 1 kW/cm² to 1 GW/cm². Unfortunately, such intense illumination often causes severe phototoxicity in living cells and photobleaching of fluorescent labels, thereby limiting the widespread application of super-resolution imaging in the life sciences. To address this issue, we previously developed a super-resolution imaging method, SPoD-OnSPAN (super-resolution polarization demodulation/on-state polarization angle narrowing), which can be performed under extremely low illumination power densities. SPoD-OnSPAN enables super-resolution imaging at power densities as low as 1 W/cm², thereby offering excellent biocompatibility. More recently, we further extended SPoD-OnSPAN to two-color imaging, making it possible to visualize two distinct intracellular components at super-resolution. In this chapter, we describe the methodology for two-color SPoD-OnSPAN imaging of doubly labeled cells, including the instrumentation, sample preparation, and imaging procedures.