Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed dual-wedge prisms

Single-molecule localization microscopy (SMLM) enables super-resolution imaging on conventional fluorescent microscopes. Spectroscopic SMLM (sSMLM) further allows highly multiplexed super-resolution imaging. We report an easy-to-implement symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM design that maximizes photon utilization. We first symmetrically dispersed photons to the −1 st and +1 st orders in an optical assembly using two identical dual-wedge prisms (DWPs). Then we computationally extracted the fluorophores’ spatial position and spectral characteristics using photons in both the −1 st and +1 st orders. Theoretical analysis and experimental validation showed lateral and spectral precisions of 10.1 nm and 0.3 nm, respectively, representing improvements of 28% and 48% over our previous DWP-based system, where emitted photons are divided separately for spatial and spectral analyses.