Single-particle tracking (SPT) has enabled direct observations of particle dynamics with sub-millisecond temporal resolution and submicron spatial resolution inside complex biological systems. Molecular trafficking within cells, tissues, and engineered 3D multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. To achieve deep and high-resolution 3D tracking, we have developed a two-photon, 3D single-particle tracking (2P-3D-SPT) method capable of tracking particles at depths up to 200 μm in scattering samples with 22/90 [xy/z] nm spatial localization precision and 50 µs temporal resolution. At shallow depths, the localization precision can be as good as 35 nm in all three dimensions. We then demonstrate tracking of EGFR (Epidermal Growth Factor Receptor) complexes tagged with fluorescent beads in tumor spheroids, demonstrating deep 3D SPT in multicellular models. More details can be found in Liu, ACS Nano, 2020.
