Method of Synthetic Motion for Testing Single Particle Tracking Microscopes

Abstract

The study of cells and molecules at the nanometer scale necessitates the development of methods that can localize and track single particles, collectively known as single particle tracking microscopes (SPTM). However successful these techniques are, it is difficult to compare them directly, especially as one is most interested in their performance on a specific experimental setup. In this work, we demonstrate the use of synthetic motion (SM) as a basis for testing, calibrating, optimizing, and comparing SPTM techniques. SM, first described by Michael Saxton, is a method of moving a fixed fluorophore along a trajectory, by a stochastic motion model realization. This allows a single particle to move with a known ground-truth in the acquired image of an SPTM, allowing for comparison to the estimated positions, trajectory, and motion model parameters. Having the ground truth then allows for detailed and precise experimental analysis and comparison of specific SPTMs with very different detection and signal processing. However, when implementing SM, one must take into account the physical limitations of the actuation modality used to achieve motion on the microscope. Piezo-actuated microscope stages make a great platform for SM due to its large actuation range, precise positioning, and high speeds. However, achieving accurate and precise positioning requires overcoming several challenges that arise due to the nature of the piezo-actuators. The effects of controller quantization, actuator slew rate, amplifier input slew rate, and bandwidth limitations on SM are analyzed. Additionally, a feedforward-feedback control system can increase the bandwidth of the stage while maintaining the precise and accurate motion needed for the fast acquisition rates of modern SPTMs and for producing intraframe motion-blur. With these challenges overcome, SM becomes a powerful tool for the experimental analysis of SPT algorithms, hardware, and microscope instrument designs.