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Single-molecule fluorescence resonance energy transfer (smFRET) remains a widely utilized and powerful tool for quantifying heterogeneous interactions and conformational dynamics of biomolecules. However, traditional smFRET experiments either are limited to short observation times (typically less than 1 ms) in the case of "burst" confocal measurements or require surface immobilization which usually has a temporal resolution limited by the camera framing rate. We developed a smFRET 3D tracking microscope that is capable of observing single particles for extended periods of time with high temporal resolution. The confocal tracking microscope utilizes closed-loop feedback to follow the particle in solution by recentering it within two overlapping tetrahedral detection elements, corresponding to donor and acceptor channels. We demonstrated the microscope's multicolor tracking capability via random walk simulations and experimental tracking of 200 nm fluorescent beads in water with a range of apparent smFRET efficiency values, 0.45-0.69. We also demonstrated the microscope's capability to track and quantify double-stranded DNA undergoing intramolecular smFRET in a viscous glycerol solution. In future experiments, the smFRET 3D tracking system will be used to study protein conformational dynamics while diffusing in solution and native biological environments with high temporal resolution.

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.

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.