Orthogonally Dispersed Spectroscopic Single-Molecule Localization Microscopy.

Area of Science:

• Biophysics
• Optical Microscopy
• Spectroscopy

Background:

• Spectroscopic single-molecule localization microscopy (sSMLM) enables simultaneous spatial and spectral data acquisition.
• A key challenge in sSMLM is the photon budget limitation, forcing a compromise between localization and spectral performance.
• Existing methods often suffer from dispersion-dependent artifacts affecting localization precision.

Purpose of the Study:

• To introduce orthogonally dispersed sSMLM (ODsSMLM) to overcome the photon budget dilemma in sSMLM.
• To enable the use of all photons for both localization and spectral characterization.
• To achieve isotropic lateral localization precision and reduce artifacts.

Main Methods:

• Modulating single-molecule emission spectra using an orthogonal structure.
• Implementing ODsSMLM for simultaneous spatial and spectral data acquisition.
• Validating the method with simulated data and dual-color imaging experiments.

Main Results:

• ODsSMLM utilizes all photons for both localization and spectral analysis.
• Simulated data showed 10 nm localization and 1.1 nm spectral precision with a 3000-photon budget.
• Dual-color imaging of microtubules and clathrin achieved 27 nm isotropic lateral resolution.

Conclusions:

• ODsSMLM effectively resolves the trade-off between localization and spectral precision in sSMLM.
• The method provides isotropic lateral localization, eliminating dispersion-dependent artifacts.
• ODsSMLM offers a significant advancement for multicolor imaging and molecular characterization.