Balancing performance and complexity of dual-wedge prism-based spectroscopic single-molecule localization microscopy

Spectroscopic single-molecule localization microscopy (sSMLM) enables the imaging of multiple fluorophores with high spatial and spectral resolution. One popular DWP-based approach is the dual-wedge prism (DWP)-based sSMLM, which provides a compact and alignment-stable method for spectral dispersion. However, the trade-offs between localization precision, spectral precision, and experimental complexity are not well understood.

This study systematically compares five different DWP-based sSMLM configurations, including both two-dimensional (2D) and three-dimensional (3D) setups. The configurations include single DWP (DWP-sSMLM) and symmetrically-dispersed DWP (SDDWP-sSMLM) setups. The researchers evaluated three key factors: lateral precision, spectral precision, and ease of use.

The results show that SDDWP configurations acquire spectral images in both channels, utilizing both for spatial localization. This approach yields the highest lateral and spectral precision among the tested setups. However, for applications that do not require axial information, the 2D-DWP configuration offers a simple and straightforward solution with robust performance.

The study provides a guideline for selecting the most appropriate DWP configuration based on the specific experimental needs, balancing the trade-offs between performance and complexity.