High-speed, real-time feedback-driven single particle tracking with concurrent smFRET - PROJECT SUMMARY: The goal of this project is to establish proof-of-concept for a new high speed, Real-Time Feedback-Driven Single Particle Tracking (RT-FD-SPT) fluorescence microscope with concurrent spectroscopic readout. The project is motivated in large part by open questions in the spatiotemporal dynamics of activated growth factors. The ability to follow individual growth factors moving in their natural environment with high spatial and temporal resolution while simultaneously gathering information about the internal state of the tracked molecule promises an improved understanding of growth factor regulatory mechanisms that promote tissue development and of regulatory breakdown with pathology. The ability to detect and observe the dynamic behavior of individual molecules has been revolutionary in the life sciences. RT-FD-SPT methods are an emerging class of techniques that offer higher temporal resolution and an extended tracking range in all three dimensions when compared to imaging-based methods. Existing RT-FD-SPT instruments, however are extremely limited in the speed of tracking that can be achieved, in the duration of tracking, or both. Moreover, the ability to do simultaneous single molecule spectroscopy is largely underdeveloped despite its promise for studying, for example, structural changes in a molecule as it interacts with its environment. We will create a novel tracking approach that leverages dual stage scanners that create motion in each axis through a combination of a short range, high bandwidth actuator in series with a long range, lower bandwidth one. Such a system can realize high speed, high precision motion over the ranges necessary for tracking a molecule over tens of microns. These dual stage scanners will be combined with new nonlinear controllers that directly operate on measured photon counts to realize particle tracking, allowing for the very high sample rate feedback control necessary to track fast moving particles. The proposed structure also naturally enables the inclusion of a secondary excitation source for concurrent spectroscopy, which in this project takes the form of Alternating laser EXcitation (ALEX)-based single molecule FRET. If this exploratory project can establish feasibility of the approach, it will lay the foundation for future work that includes real-time adaptive shaping of the excitation beam and adaptation of the controller parameters to overcome the tradeoff between signal intensity, tracking quality, and tracking duration, and validation in model growth factor systems. If successful, the new instrument will significantly expand our ability to investigate single molecule dynamics and look at causes of pathology at the molecular level.
