Abstract
Significance: 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. Aim: We developed a Monte Carlo (MC)-based model that predicts the imaging output of the DWP-based sSMLM system given different system parameters. Approach: 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. Results: Our 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. Conclusions: 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.
| Original language | English (US) |
|---|---|
| Article number | S11502 |
| Journal | Journal of Biomedical Optics |
| Volume | 29 |
| DOIs | |
| State | Published - Jan 15 2024 |
Funding
The authors sincerely acknowledge the generous support from the National Institutes of Health (NIH; Grant Nos. R01GM139151, R01GM140478, U54CA268084, and R01GM143397) and the National Science Foundation (NSF; Grant Nos. CHE-1954430 and EFMA-1830969). This work used the EPIC facility of Northwestern University's Atomic and Nanoscale Characterization Experimental Center (NUANCE), which has received support from the SHyNE Resource (Grant No. NSF ECCS-2025633), the IIN, and Northwestern's MRSEC Program (Grant No. NSF DMR-1720139). Wei-Hong Yeo is supported by the Christine Enroth-Cugell Fellowship for Vision and Neuroscience at Northwestern University. The authors sincerely acknowledge the generous support from the National Institutes of Health (NIH; Grant Nos. R01GM139151, R01GM140478, U54CA268084, and R01GM143397) and the National Science Foundation (NSF; Grant Nos. CHE-1954430 and EFMA-1830969). This work used the EPIC facility of Northwestern University\u2019s Atomic and Nanoscale Characterization Experimental Center (NUANCE), which has received support from the SHyNE Resource (Grant No. NSF ECCS-2025633), the IIN, and Northwestern\u2019s MRSEC Program (Grant No. NSF DMR-1720139). Wei-Hong Yeo is supported by the Christine Enroth-Cugell Fellowship for Vision and Neuroscience at Northwestern University.
Keywords
- Monte Carlo simulation
- single-molecule localization microscopy
- spectroscopy
- super-resolution microscopy
ASJC Scopus subject areas
- Electronic, Optical and Magnetic Materials
- Biomaterials
- Atomic and Molecular Physics, and Optics
- Biomedical Engineering