Real-time 3D single particle tracking in noisy environment using lifetime-gated two-photon emission

      The real-time 3D single-particle tracking technique offers great potentials to reveal the behaviors of the single units in different environments. However, intrinsic background photons and excitation-induced scatterings from the complex environments render observation of the behaviors of the probes difficult. A microscopy platform that leverages the arrival time of individual photons to enable 3D single-particle tracking of fast-moving (translational diffusion coefficient of ≃ 3.3 μm²/s) particles in high-background environments is presented here. It combines a hardware-based time-gating module, which enables the rate of photon processing to be as high as 100 MHz, with a two-photon excited 3D single-particle tracking confocal microscope to enable high sample penetration depth. Proof-of-principle experiments, where single giant quantum dots (gQDs) were tracked in solutions containing dye-stained cellulose with a ~100-fold improvement in signal-to-background ratio using the hardware-based time-gating module, are shown here. Compared with previous works, a 33-fold improvement was achieved in terms of the mobility of the particles that can be tracked. This implementation of a time-gated real-time 3D single-particle tracking capacity, which should in the future be able to be coupled to other imaging and control modalities, can be expected to broaden the scope of experimentally accessible systems. Such a microscope design is anticipated to be of use to a variety of communities who wish to track single-particles in cellular environments, which commonly have high fluorescence and a scattering background.