Advancing 3D Single Molecule Tracking by Time-Gating and Fast Simultaneous Spinning Disk Imaging for Contextual Information

Abstract

Three dimensional single molecule tracking is an essential technique for investigating biomolecular trafficking and signaling. Here we report the latest improvements to our confocal based tracking system. First, we added a Nipkow spinning disk imaging system. This system takes images of the plane of the tracked molecule, thus yielding contextual information about the molecule's environment. Laser illumination is used for the spinning disk system which together with an acousto-optic modulator allows for exposure times of 10 to 50 ms, thus greatly minimizing motion blur effects due to the feedback motion during tracking. Second, we introduced real-time photon time-gating to our tracking setup which allows tracking using only those photons detected within a defined time-window relative to the excitation laser pulse. Gating out detected photons within the first few nanoseconds after the excitation laser pulse greatly reduces Raman scattering and short lived fluorescence, and significantly increases the signal-to-noise ratio for tracked molecules with a long fluorescence lifetime. We also present a model for choosing the optimal time-gate based upon the fluorescence count rates and life times of the background and the tracked molecule.