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 by analyzing individual fluorescence emissions.
• Spectroscopic SMLM (sSMLM) adds spectral data but faces precision loss due to photon splitting.

Purpose of the Study:

• To present a novel symmetrically dispersed dual-wedge prism (SDDWP)-sSMLM system.
• To maximize photon utilization for simultaneous spatial and spectral analysis in SMLM.
• To improve precision and enable multiplexed imaging and high-concentration nanoparticle tracking.

Main Methods:

• Utilized a symmetrically dispersed dual-wedge prism (SDDWP) system for equal photon splitting and symmetric dispersion.
• Employed computational extraction of spatial positions and spectra from all collected photons.
• Integrated the system into a compact module for easy upgrade from existing SMLM setups.

Main Results:

• Achieved a 27% improvement in spatial precision and a 48% improvement in spectral precision compared to previous sSMLM systems.
• Demonstrated multiplexed imaging of cellular structures (peroxisomes, microtubules, mitochondria) using spectrally overlapping dyes with a single laser.
• Enabled massively parallel tracking of spectrally tagged nanoparticles at five times higher concentrations than previously possible.

Conclusions:

• The SDDWP-sSMLM system significantly enhances precision in both spatial and spectral dimensions.
• This technology facilitates advanced multiplexed imaging and high-density nanoparticle tracking.
• The compact, upgradeable design promotes broader adoption of advanced spectroscopic SMLM techniques.