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

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. Access through your institution Buy or subscribe This

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OF FOURIER PTYCHOGRAPHY Article 10 February 2021 LENS-FREE ON-CHIP 3D MICROSCOPY BASED ON WAVELENGTH-SCANNING FOURIER PTYCHOGRAPHIC DIFFRACTION TOMOGRAPHY Article Open access 05 September

2024 DESIGN, ASSEMBLY, ALIGNMENT AND APPLICATION OF A VERSATILE, OPEN-SOURCE, SINGLE-PIXEL MICROSCOPE Article Open access 22 May 2025 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

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Song, P. et al. Freeform illuminator for computational microscopy. _Intell. Comput._ 2, 0015 (2023). Article  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * These authors

contributed equally: Shaowei Jiang, Pengming Song, Tianbo Wang. AUTHORS AND AFFILIATIONS * 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 * Hangzhou Institute of Technology, Xidian University, Hangzhou, China Chengfei Guo *

Department of Electronic and Electrical Engineering, University of Sheffield, Sheffield, UK Andrew Maiden * Diamond Light Source, Harwell Science and Innovation Campus, Chilton, UK Andrew

Maiden Authors * Shaowei Jiang View author publications You can also search for this author inPubMed Google Scholar * Pengming Song View author publications You can also search for this

author inPubMed Google Scholar * Tianbo Wang View author publications You can also search for this author inPubMed Google Scholar * Liming Yang View author publications You can also search

for this author inPubMed Google Scholar * Ruihai Wang View author publications You can also search for this author inPubMed Google Scholar * Chengfei Guo View author publications You can

also search for this author inPubMed Google Scholar * Bin Feng View author publications You can also search for this author inPubMed Google Scholar * Andrew Maiden View author publications

You can also search for this author inPubMed Google Scholar * Guoan Zheng View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS 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). PEER REVIEW PEER REVIEW INFORMATION _Nature Protocols_ thanks Zhengjun Liu, Fucai Zhang and the other,

anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. 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. RIGHTS

AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 06 September 2022 * Accepted: 03 March 2023 * Published: 29 May 2023 * Issue Date: July 2023 * DOI:

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