Abstract

The dual-wedge prism (DWP)-based spectroscopic single-molecule localization microscopy (sSMLM) system offers improved localization precision and adjustable spectral or localization performance, but its nonlinear spectral dispersion presents a challenge. A systematic method can help understand the challenges and thereafter optimize the DWP system’s performance by customizing the system parameters to maximize the spectral or localization performance for various molecular labels. We developed a Monte Carlo (MC)-based model that predicts the imaging output of the DWP-based sSMLM system given different system parameters. We assessed our MC model’s localization and spectral precisions by comparing our simulation against theoretical equations and fluorescent microspheres. Furthermore, we simulated the DWP-based system using beamsplitters (BSs) with a reflectance (R):transmittance (T) of R50:T50 and R30:T70 and their tradeoffs. The MC model accurately predicted the localization precision, spectral precision, spectral peaks, and spectral widths of fluorescent microspheres, as validated by experimental data. Our work enhances the theoretical understanding of DWP-based sSMLM for multiplexed imaging, enabling performance optimization.