Three-dimensional single particle tracking using 4π self-interference of temporally phase-shifted fluorescence

Single particle tracking in three dimensions is an indispensable tool for studying dynamic processes in various disciplines, including material sciences, physics, and biology, but often shows anisotropic three-dimensional spatial localization precision, which restricts the tracking precision, and/or a limited number of particles that can be tracked simultaneously over extended volumes. Here we developed an interferometric, three-dimensional fluorescence single particle tracking method based on conventional widefield excitation and temporal phase-shift interference of the emitted, high-aperture-angle, fluorescence wavefronts in a greatly simplified, free-running, triangle interferometer that enables tracking of multiple particles at the same time with <10-nm spatial localization precision in all three dimensions over extended volumes (~35 × 35 × 2 μm³) at video rate (25 Hz). We applied our method to characterize the microenvironment of living cells and up to ~40 μm deep in soft materials.