SignificanceThe 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.AimWe developed a Monte Carlo (MC)-based model that predicts the imaging output of the DWP-based sSMLM system given different system parameters.ApproachWe 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.ResultsOur MC simulation showed average deviations of 2.5 and 2.1 nm for localization and spectral precisions against theoretical equations and 2.3 and 1.0 nm against fluorescent microspheres. An R30:T70 BS improved the spectral precision by 8% but worsened the localization precision by 35% on average compared with an R50:T50 BS.ConclusionsThe 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.

中文翻译：

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基于双楔棱镜的光谱单分子定位显微镜的物理信息蒙特卡罗模拟

意义基于双楔形棱镜（DWP）的光谱单分子定位显微镜（sSMLM）系统提供了更高的定位精度和可调节的光谱或定位性能，但其非线性光谱色散提出了挑战。系统方法可以帮助理解挑战，然后通过定制系统参数来优化 DWP 系统的性能，以最大限度地提高各种分子标签的光谱或定位性能。目的我们开发了一种基于蒙特卡罗 (MC) 的模型，可以预测 DWP 系统的成像输出给定不同系统参数的基于 DWP 的 sSMLM 系统。方法我们通过将我们的模拟与理论方程和荧光微球进行比较来评估我们的 MC 模型的定位和光谱精度。此外，我们使用反射率 (R):透射率 (T) 为 R50:T50 和 R30:T70 的分束器 (BS) 模拟基于 DWP 的系统及其权衡。结果我们的 MC 模拟显示定位的平均偏差为 2.5 和 2.1 nm以及针对理论方程的光谱精度以及针对荧光微球的 2.3 和 1.0 nm 的光谱精度。与R50:T50 BS相比，R30:T70 BS的光谱精度提高了8%，但定位精度平均下降了35%。结论MC模型准确预测了荧光的定位精度、光谱精度、光谱峰值和光谱宽度微球，经实验数据验证。我们的工作增强了对用于多重成像的基于 DWP 的 sSMLM 的理论理解，从而实现性能优化。