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.

Grant Number: 1R21GM157695-01
NIH Institute/Center: NIH
Principal Investigator: Sean Andersson