Three-dimensional tracking of multiple particles in large depth of field using dual-objective bifocal plane imaging

Tracking moving particles in cells by single particle tracking is an important optical approach widely used in biological research. In order to track multiple particles within a whole cell simultaneously, a parallel tracking approach with large depth of field was put forward. It was based on distorted grating and dual-objective bifocal imaging, making use of the distorted grating to expand the depth of field, dual-objective to gather as many photons as possible, and bifocal plane imaging to realize three-dimensional localization. Simulation of parallel tracking of two particles moving along the z axis demonstrated that even when the two are axially separated by 10 μm, they can both be localized simultaneously with transversal precision better than 5 nm and axial precision better than 20 nm.