Two-photon real-time device for single-particle holographic tracking (red shot)

TL;DR

This paper introduces a real-time, high-precision 3D tracking method for single particles using two-photon holography and nonlinear nanoparticles, enabling detailed biological transport studies.

Contribution

It demonstrates a novel two-photon holographic tracking system with nanometer precision and high refresh rates, suitable for observing fast intracellular particle dynamics.

Findings

Achieved 15 nm localization precision at 1.1 kHz refresh rate.

Tracked particles moving faster than 30 μm/s.

Successfully monitored intracellular transport in neurites.

Abstract

Three-dimension real-time tracking of single emitters is an emerging tool for assessment of biological behavior as intraneuronal transport, for which spatiotemporal resolution is crucial to understand the microscopic interactions between molecular motors. We report the use of second harmonic signal from nonlinear nanoparticles to localize them in a super-localization regime, down to 15 nm precision, and at high refreshing rates, up to 1.1 kHz, allowing us to track the particles in real-time. Holograms dynamically displayed on a digital micro-mirror device are used to steer the excitation laser focus in 3D around the particle on a specific pattern. The particle position is inferred from the collected intensities using a maximum likelihood approach. The holograms are also used to compensate for optical aberrations of the optical system. We report tracking of particles moving faster than 30 $\mu$m/s with an uncertainty on the localization around 40 nm. We have been able to track freely moving particles over tens of micrometers, and directional intracellular transport in neurites.