Theoretical comparison of real-time feedback-driven single-particle tracking techniques.

Area of Science:

• Biophysics
• Spectroscopy
• Nanotechnology

Background:

• Real-time feedback-driven single-particle tracking (RT-FD-SPT) enables single-molecule spectroscopy of freely diffusing particles.
• Existing comparisons of RT-FD-SPT methods are limited by variations in samples and experimental setups.

Purpose of the Study:

• To directly compare the performance of different RT-FD-SPT methods using statistical calculations and dynamical simulations.
• To evaluate the trade-offs between speed and precision for various tracking techniques.
• To compare the efficacy of fluorescence-based and interferometric scattering (iSCAT) approaches for particle tracking.

Main Methods:

• Statistical calculations and dynamical simulations were employed for direct method comparison.
• Evaluated methods include the orbital method, knight's tour (grid scan), and MINFLUX.
• Both fluorescence and interferometric scattering (iSCAT) detection methods were considered.

Main Results:

• A fundamental trade-off exists between tracking speed and precision; knight's tour offers speed, while MINFLUX provides precision.
• The performance of iSCAT versus fluorescence depends on particle size, density, and photophysical properties.
• iSCAT generally offers superior tracking, especially when using a non-absorbed wavelength for illumination.

Conclusions:

• This work elucidates fundamental performance aspects of RT-FD-SPT methods.
• The findings will aid researchers in selecting the optimal RT-FD-SPT method for specific applications.
• The simulation approach can be extended to evaluate other RT-FD-SPT techniques.