High-speed, real-time feedback-driven single particle tracking with concurrent smFRET

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. This instrument will allow following 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. The project will create a novel tracking approach leveraging dual stage scanners that combine short-range, high-bandwidth actuators with long-range, lower-bandwidth ones to achieve high speed, high precision motion over tens of microns. These scanners will be integrated with new nonlinear controllers operating on measured photon counts for high sample rate feedback control. The system will also enable concurrent spectroscopy, specifically Alternating laser EXcitation (ALEX)-based single molecule FRET. The exploratory project aims to establish feasibility, laying the foundation for future work including real-time adaptive shaping of the excitation beam, adaptation of controller parameters, and validation in model growth factor systems.