Physically informed Monte Carlo simulation of dual-wedge prism-based spectroscopic single-molecule localization...

Area of Science:

• Optics and Photonics
• Biomedical Imaging
• Super-resolution Microscopy

Background:

• The dual-wedge prism (DWP) system enhances spectroscopic single-molecule localization microscopy (sSMLM) but faces challenges due to nonlinear spectral dispersion.
• Optimizing DWP systems requires understanding parameter effects on spectral and localization performance for diverse molecular labels.

Purpose of the Study:

• To develop a predictive model for DWP-based sSMLM imaging performance.
• To systematically analyze the impact of system parameters on localization and spectral precision.

Main Methods:

• A Monte Carlo (MC) based model was developed to simulate DWP-based sSMLM imaging output.
• The MC model's accuracy was validated against theoretical equations and experimental data from fluorescent microspheres.
• Simulations explored the trade-offs using beamsplitters with different reflectance:transmittance ratios (R50:T50 and R30:T70).

Main Results:

• The MC model demonstrated high accuracy, with average deviations of 2.5 nm (localization) and 2.1 nm (spectral) compared to theory.
• Simulations against fluorescent microspheres showed even lower deviations: 2.3 nm (localization) and 1.0 nm (spectral).
• A R30:T70 beamsplitter improved spectral precision by 8% at the cost of a 35% decrease in localization precision compared to R50:T50.

Conclusions:

• The MC model accurately predicts key performance metrics of DWP-based sSMLM, including localization and spectral precision.
• This work provides a validated tool for understanding and optimizing DWP systems for multiplexed imaging applications.
• The findings facilitate customized system parameter adjustments to maximize performance for specific molecular labels.