Long axial range 3D single-particle tracking using birefringent substrates.

3D single-particle tracking is a critical imaging technique for visualizing molecular motion in complex environments, including biological cells. Expanding the trackable depth of the 3D tracking technique to a greater range would broaden its applicability to larger biological samples. Most high-throughput 3D tracking techniques rely on the engineering of the point spread function of the optical system to precisely determine the 3D coordinate of the particle using spatial light modulators. Here, we report 3D single-particle tracking using a birefringent material, mica, as a substrate for mounting a sample. The spatial pattern of the fluorescence emitted by fluorescent nanoparticles captured at the image plane shows an axial position dependence over the tens of micrometers range due to the birefringent characteristic of the mica substrate, enabling us to localize the emitter with an accuracy better than 30 nm over an axial range of 30 µm. We demonstrate that our 3D tracking method can simultaneously track multiple particles separated by a 30 µm distance in the axial axis. We further validate our 3D tracking applicability in plant cells, which are significantly larger than animal cells. This work contributes to advancing single-particle 3D tracking using birefringent substrates with unique optical characteristics.