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

Abstract

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.