Three-dimensional particle tracking using a dual-objective fluorescent reflection system with spherical aberration

It is often difficult to implement complex microscopy systems without spherical aberration. Herein, we developed a novel, robust, three-dimensional (3D), bifocal plane, single-particle tracking technique, based on a dual-objective fluorescent reflection system with spherical aberration (DOFR–SA). It can simultaneously image a pair of focused and defocused planes containing fluorescent particles with a single camera instead of splitting photons into two channels. Based on the 3D DOFR–SA, the desired position accuracy along the z-axis was achieved without compromising the precisions of the (x, y ) positions, even with limited number of photons from a single molecule. Accordingly, this method was applied to fluorescent particle tracking in biofluids and living cells with high-spatial and temporal precisions.