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Spatial- and Fourier-domain ptychography for high-throughput bio-imaging

Nature Protocols volume 18, pages 2051–2083 (2023)Cite this article

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Abstract

First envisioned for determining crystalline structures, ptychography has become a useful imaging tool for microscopists. However, ptychography remains underused by biomedical researchers due to its limited resolution and throughput in the visible light regime. Recent developments of spatial- and Fourier-domain ptychography have successfully addressed these issues and now offer the potential for high-resolution, high-throughput optical imaging with minimal hardware modifications to existing microscopy setups, often providing an excellent trade-off between resolution and field of view inherent to conventional imaging systems, giving biomedical researchers the best of both worlds. Here, we provide extensive information to enable the implementation of ptychography by biomedical researchers in the visible light regime. We first discuss the intrinsic connections between spatial-domain coded ptychography and Fourier ptychography. A step-by-step guide then provides the user instructions for developing both systems with practical examples. In the spatial-domain implementation, we explain how a large-scale, high-performance blood-cell lens can be made at negligible expense. In the Fourier-domain implementation, we explain how adding a low-cost light source to a regular microscope can improve the resolution beyond the limit of the objective lens. The turnkey operation of these setups is suitable for use by professional research laboratories, as well as citizen scientists. Users with basic experience in optics and programming can build the setups within a week. The do-it-yourself nature of the setups also allows these procedures to be implemented in laboratory courses related to Fourier optics, biomedical instrumentation, digital image processing, robotics and capstone projects.

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Fig. 1: The modern form of ptychography.
Fig. 2: Spatial-domain CP and FP.
Fig. 3: Overview of Procedures 1 and 2 for developing the FP and CP platforms.
Fig. 4: Incident angle calibration and alignment for FP.
Fig. 5: Hardware implementation of FP.
Fig. 6: Reconstruction process of FP.
Fig. 7: Hardware implementation of CP.
Fig. 8: Tracking the motion of the coded sensor in CP.
Fig. 9: Reconstruction process of CP.
Fig. 10: Stage assembling process for CP.
Fig. 11: System integration for CP.
Fig. 12: Troubleshooting for FP.
Fig. 13: Troubleshooting for CP.
Fig. 14: Reconstructions with precalibrated ptychographic probes versus blind reconstructions.
Fig. 15: Representative imaging results using FP.
Fig. 16: Imaging fixed biospecimens using CP.
Fig. 17: Time-lapse monitoring of live cells using lensless CP.

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Data availability

The main data supporting this study are available within the article, Supplementary Data and the primary supporting study10,11,14. Experimental datasets for both setups in this study are available in Zenodo: https://doi.org/10.5281/zenodo.7492626.

Code availability

All related MATLAB and Arduino code is provided in Supplementary Software. Additional code for testing experimental datasets is available in Zenodo: https://doi.org/10.5281/zenodo.7492626.

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Acknowledgements

We thank Z. Bian and A. Pirhanov for their assistance in sample preparation. This work was partially supported by the UConn SPARK grant, UConn Research Excellence Program, National Science Foundation award 2012140 and National Institute of Health award U01-NS113873. P.S. also acknowledges the support of the Thermo Fisher Scientific Fellowship.

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Author notes

  1. These authors contributed equally: Shaowei Jiang, Pengming Song, Tianbo Wang.

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Connecticut, Storrs, USA

    Shaowei Jiang, Pengming Song, Tianbo Wang, Liming Yang, Ruihai Wang, Chengfei Guo, Bin Feng & Guoan Zheng

  2. Hangzhou Institute of Technology, Xidian University, Hangzhou, China

    Chengfei Guo

  3. Department of Electronic and Electrical Engineering, University of Sheffield, Sheffield, UK

    Andrew Maiden

  4. Diamond Light Source, Harwell Science and Innovation Campus, Chilton, UK

    Andrew Maiden

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Contributions

G.Z. conceived the project. S.J., P.S. and G.Z. designed the pipeline. S.J., P.S., T.W. and G.Z. developed the prototype systems and prepared the display items. S.J., P.S., T.W. and L.Y. developed the data acquisition and processing pipelines for the protocol. T.W. and C.G. prepared all SolidWorks design files for the protocols. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Guoan Zheng.

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Competing interests

G.Z. is a named inventor on the following patents related to Fourier ptychography (US Patent, nos. 9,817,224, 9,864,184, 9,497,379) and coded ptychography (US Patent, no. 11,487,099).

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Nature Protocols thanks Zhengjun Liu, Fucai Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Zheng, G. et al. Nat. Photonics 7, 739-745 (2013): https://doi.org/10.1038/nphoton.2013.187

Jiang, S. et al. ACS Photonics 8, 3261-3271 (2021): https://doi.org/10.1021/acsphotonics.1c01085

Jiang, S. et al. Biosens. Bioelectron. 196, 113699 (2022): https://doi.org/10.1016/j.bios.2021.113699

Supplementary information

Supplementary Information

Supplementary Figs. 1–4.

Reporting Summary

Supplementary Software 1

MATLAB code and Arduino code for FP and CP.

Supplementary Data 1

SolidWorks design files for FP and CP.

Supplementary Video 1

Operation of the FP platform.

Supplementary Video 2

Operation of the CP platform.

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Jiang, S., Song, P., Wang, T. et al. Spatial- and Fourier-domain ptychography for high-throughput bio-imaging. Nat Protoc 18, 2051–2083 (2023). https://doi.org/10.1038/s41596-023-00829-4

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