No 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.