Maximizing photon utilization in spectroscopic single-molecule localization microscopy using symmetrically dispersed...

Area of Science:

• Optical Microscopy
• Super-Resolution Imaging
• Spectroscopy

Background:

• Single-molecule localization microscopy (SMLM) provides super-resolution capabilities on standard fluorescent microscopes.
• Spectroscopic SMLM (sSMLM) enables highly multiplexed imaging by analyzing spectral information alongside spatial localization.

Purpose of the Study:

• To develop an easy-to-implement sSMLM design that maximizes photon utilization.
• To improve the precision of spatial localization and spectral characterization in sSMLM.
• To enhance the capabilities of super-resolution microscopy through advanced optical design.

Main Methods:

• Implementation of a symmetrically dispersed dual-wedge prism (SDDWP) optical assembly.
• Symmetrical dispersion of emitted photons into -1st and +1st orders using two identical DWPs.
• Computational extraction of fluorophore spatial position and spectral characteristics from dispersed photons.

Main Results:

• Achieved lateral precision of 10.1 nm and spectral precision of 0.3 nm.
• Demonstrated a 28% improvement in lateral precision compared to previous DWP systems.
• Showcased a 48% improvement in spectral precision compared to previous DWP systems.

Conclusions:

• The SDDWP-sSMLM design offers a significant advancement in SMLM technology.
• This method enhances multiplexing capabilities and imaging accuracy in super-resolution microscopy.
• The improved precision facilitates more detailed biological investigations at the nanoscale.