Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed...

Single-molecule localization microscopy (SMLM) achieves super-resolution by analyzing individual fluorescence emissions. Spectroscopic SMLM (sSMLM) extends this by capturing spectral data but suffers from reduced precision due to photon splitting. We present a symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM system that maximizes photon use for both spatial and spectral analyses. Fluorescence photons are equally split and symmetrically dispersed using two identical dual-wedge prisms, with spatial positions and spectra computationally extracted using all collected photons. This approach improves spatial and spectral precisions by 27% and 48%, respectively, over prior sSMLM systems. Using a single excitation laser, we achieve multiplexed imaging of peroxisomes, microtubules, and mitochondria labeled with spectrally overlapping dyes (DY-634, AF647, CF660C). We also demonstrate massively parallel tracking of spectrally tagged nanoparticles at concentrations five times higher than previously reported. The entire system is integrated into a compact module, facilitating upgrade from an existing SMLM system to a spectroscopic SMLM.