Two-photon single particle tracking in 3-D

Transport processes are important in biology and medicine. Examples include virus docking and infection, endocytosis of extracellular protein and phagtocytosis of antigenic material. Trafficking driven by molecular motors inside a complex three dimensional environment is a shared common theme. The complex sequence of these events are difficult to resolve with conventional techniques where the action of many cells are asynchronously averaged. Single particle tracking (SPT) was developed by Ghosh and Webb to address this problem and has proven to be a powerful technique in understanding membrane-protein interaction. Since the traditional SPT method uses wide field illumination and area detectors, it is limited to the study of two-dimensional systems. In this presentation we report the development of a 3-D single particle tracking technique using two-photon excitation. Using a real-time feedback system, we can dynamically position the sub-femtoliter two-photon excitation volume to follow the fluorescent particle under transport by maximizing the detected fluorescent intensity. Further, fluorescence spectroscopy can be performed in real time along the particle trajectory to monitor the underlying biochemical signals driving this transport process. The first application of this instrument will focus on the study of antigen endocytosis process of macrophages.