Active Feedback 3D Single-Molecule Tracking

Abstract

Single molecule tracking has the potential to revolutionize the study of biological systems. However, most current single molecule imaging and tracking methods are limited to two dimensions. Meanwhile, real-time 3D single molecule tracking methods, which use active feedback to "lock-on" to single molecules in solution have until now been limited to observation times of 500 msec or less, greatly limiting their utility. Recently we developed a real-time 3D single particle tracking method known as 3D Dynamic Photon Localization Tracking (3D-DyPLoT). 3D-DyPLoT dynamically scans a focused laser spot in 3D with a tunable acoustic gradient lens and a 2D electro-optic deflector and has demonstrated real-time tracking at high diffusive speeds (up to 20 µm2/s) and low photon count rates 10 kHz. Here we demonstrate that the high tracking speed, sensitivity, and robustness of 3D-DyPLoT make it ideally suited active feedback 3D single molecule tracking. By implementing optimized tracking parameters based on single molecule model simulation, 3D-DyPLoT can continuously track single fluorescent dye molecules for several minutes at a time, two orders of magnitudes longer than previously reported methods. We demonstrate the application of this new single molecule tracking microscopy to track the free, 3D diffusion of DNA, RNA and proteins in solution. With two-color observation channels, 3D-DyPLoT was also applied to observing the transcription of single DNA molecules in solution by monitoring the real-time production of mRNA. This real-time 3D single molecule tracking method promises to be powerful tool for capturing the dynamics of single biomolecules at high speeds and over 3D distances. (Supported by NIH R35GM124868 and NSF 1847899).