Molecular trafficking within engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of 15 µm. Here we present a new 3D tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 μs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built upon spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for 3D particle tracking and allows for two-photon, multi-color imaging. 3D tracking of epidermal growth factor receptor (EGFR) complexes at a depth of 100 µm in tumor spheroids is demonstrated. Our 3D tracking microscope is built upon spatiotemporally multiplexed two-photon excitation and uses time-gated analysis via a photon counting histogram to discern the molecular 3D position. Feedback control then steers the excitation to lock-on to the single molecule as it travels at a high speed. The molecular trajectories are reconstructed from the recorded actuator positions from the feedback control loop operating at 1-5 ms. Dynamics down to 50 µs can be inferred from analysis of the photon counting histogram. In our method, the first PMT channel is used for particle tracking while the second and the third PMT channels can be used for two-photon scanning microscopy, colocalization analysis, and energy transfer studies. We have coined this technique TSUNAMI (Tracking Single particles Using Nonlinear And Multiplexed Illumination).

Deep and high-resolution three-dimensional tracking of single particles using nonlinear and multiplexed illumination

251st National Meeting (2016)

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