Single-particle tracking and its application in biophysical phenotyping

Single-particle tracking (SPT) has advanced our knowledge of molecular and cellular biology since its introduction in the early 90s. Whereas important discoveries made by SPT have changed our view of the plasma membrane organization and motor protein dynamics, experimental studies of intracellular processes using SPT are rather scarce due to the lack of 3D tracking technique, trajectory analysis algorithm, and validation. This dissertation describes my work on the validation of a 3D tracking technique and the development of a trajectory analysis algorithm; the developed SPT technique and algorithm was applied to visualizing biomolecules trafficking in live cells, measuring mechanical properties of cells, monitoring DNA conformational changes, and differentiating metastatic cells from benign or less-invasive cancer cells. First, we provided an overview of current SPT technology and a detailed description of our 2-photon 3-dimensional single-particle tracking microscope termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) and improvements to TSUNAMI. Second, we applied the TSUNAMI to investigate epidermal growth factor receptor (EGFR) trafficking in single cancer cells or multicellular cancer spheroids at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 minutes and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed with 81% segment classification accuracy in the simulated movement experiments. This algorithm accurately retrieves the dynamics of EGFR trafficking from the 3D trajectories in which EGFRs travel from the plasma membrane into the deep cytosol via active transport. Third, we present an advanced version of TSUNAMI microscopy with the capability of two-color dual-particle tracking, which enables us to monitor the conformational changes of nicked or gapped double-stranded DNA and to quantify the DNA bendability in free solution with no surface interference. In the end, we develop an SPT-based biophysical phenotyping assay named Transmembrane Receptor dynamics (TReD) to differentiate metastatic cancer cells from less invasive ones. While derailed transmembrane receptor trafficking has been seen as a hallmark of tumorigenesis and increased metastatic potential of cancers, the transmembrane receptor dynamics has never been used as a physical biomarker for cancer detection. We tested a series of breast cancer and prostate cancer cell lines to validate the TReD assay and discovered the positive correlation between EGFR diffusivities and metastatic potentials. Form the instrumentation of TSUNAMI microscope and the development of trajectory analysis algorithm to their applications to the quantifications of EGFR dynamics and DNA conformational changes, we believe that the systems and methods developed in this work will provide life scientists with a powerful toolset for the future of biological research.