Abstract
SIGNIFICANCE: Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth ( z ) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field. AIM: To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system. APPROACH: We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker. RESULTS: Bead localization precision was < 25 nm in the transverse dimensions and ≤ 70 nm along the axial dimension over an axial range of 6 μm . Higher axial precision ( ≤ 50 nm ) was achieved over a shallower focal depth of 2.9 μm . 3D diffusion constants of chromatin matched expected values. CONCLUSIONS: Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.