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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 system parameters to maximize spectral or localization performance for various molecular labels.
We developed an MC-based model which 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 of Reflectance (R):Transmittance (T) of R50:T50 and R30:T70 and their tradeoffs.
Our MC simulation showed average deviations of 2.5 nm and 2.1 nm for localization and spectral precisions against theoretical equations; and 2.3 nm and 1.0 nm against fluorescent microspheres. An R30:T70 beamsplitter improved spectral precision by 8% but worsened localization precision by 35% on average compared to an R50:T50 beamsplitter.
The MC model accurately predicted 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.