All Research Areas

3D Single Particle Tracking

Molecular trafficking within cells, tissues and 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. Our aim is to develop new 3D tracking techniques and algorithms to improve 3D tracking capability in tissue and in vivo models. We have recently developed a three-dimensional tracking method, termed TSUNAMI, 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 on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolor imaging. An initial demonstration of the technique was performed on of epidermal growth factor receptor (EGFR) complexes at a depth of 100 μm in tumor spheroids. We are currently working to extend this technique’s capabilities with multi-color detection. Current application areas include early cancer diagnosis in circulating tumor cells, and study of EGFR trafficking in the cytoplasm.

Relevant Publications

Segmentation of 3D trajectories acquired by TSUNAMI microscope: An application to EGFR trafficking

Y. L. Liu, E. P. Perillo, C. Liu, P. Yu, C. K. Chou, M. C. Hung, A. K. Dunn, and H.C. Yeh, Biophysical Journal (2016)

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection

C. Liu, Y. L. Liu, E. P. Perillo, A. K. Dunn, and H. C. Yeh, IEEE Journal of Selected Topics in Quantum Electronics (2016)

Improving z­-tracking accuracy in the two-photon single-particle tracking microscope

C. Liu, Y. L. Liu, E. P. Perillo, N. Jiang, A. K. Dunn, and H. C. Yeh, Applied Physics Letters (2015)

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

E. P. Perillo, Y. L. Liu, K. Huynh, C. Liu, C. K. Chou, M. C. Hung, H. C. Yeh, and A. K. Dunn, Nature Communications (2015)
EGFR Particle Trajectory

Trajectory of a single epidermal growth factor receptor (EGFR) complex tracked for 450 seconds. The cellular features are stained, membrane (red), and nucleus (blue). Point (1) in the image denotes a period of rapid directed transport. (Perillo, et al., 2015)

3D Tracking Optical Schematic

A novel beam-splitter device is used to generate a tetrahedral focus that can "lock-on" to single fluorescent particles. The use of two-photon excitation enables the technique to be used in scattering tissue models. (Perillo, et al., 2015)