Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate

Keith D. Bonin; Sudhakar Prasad; Will Caulkins; George Holzwarth; Stephen R. Baker; Pierre-Alexandre Vidi

doi:10.1117/1.JBO.27.12.126501

29 December 2022 Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate

Keith D. Bonin, Sudhakar Prasad, Will Caulkins, George Holzwarth, Stephen R. Baker, Pierre-Alexandre Vidi

Author Affiliations +

Journal of Biomedical Optics, Vol. 27, Issue 12, 126501 (December 2022). https://doi.org/10.1117/1.JBO.27.12.126501

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.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Citation Download Citation

Keith D. Bonin, Sudhakar Prasad, Will Caulkins, George Holzwarth, Stephen R. Baker, and Pierre-Alexandre Vidi "Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate," Journal of Biomedical Optics 27(12), 126501 (29 December 2022). https://doi.org/10.1117/1.JBO.27.12.126501

Received: 3 June 2022; Accepted: 13 December 2022; Published: 29 December 2022

ACCESS THE FULL ARTICLE

JOURNAL ARTICLE
17 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY