High-fidelity tissue super-resolution imaging achieved with confocal<sup>2</sup> spinning-disk image scanning microscopy

Qianxi Liang; Wei Ren; Boya Jin; Liang Qiao; Xichuan Ge; Yunzhe Fu; Xiaoqi Lv; Meiqi Li; Peng Xi

doi:10.1038/s41377-025-01930-x

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High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy

Original Articles

- High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy
- Light: Science & Applications Vol. 14, Issue 9, Pages: 2720-2735(2025)
- Affiliations：
  
  1.Department of Biomedical Engineering, National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing, China
  2.Airy Technologies Co. Ltd, Beijing, China
  3.School of Life Sciences, Peking University, Beijing, China
- Author bio：
  
  Meiqi Li (limeiqi@pku.edu.cn)
  Peng Xi (xipeng@pku.edu.cn)
- Funds：
- DOI：10.1038/s41377-025-01930-x
  CLC：
- Received：19 February 2025，
  
  Revised：2025-06-19，
  
  Accepted：20 June 2025，
  
  Published Online：04 August 2025，
  
  Published：30 September 2025
- Accepted：
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Liang, Q. X. et al. High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy. Light: Science & Applications, 14, 2720-2735 (2025).
DOI：

Liang, Q. X. et al. High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy. Light: Science & Applications, 14, 2720-2735 (2025). DOI： 10.1038/s41377-025-01930-x.

摘要

Abstract

Super-resolution imaging has revolutionized our ability to visualize biological structures at subcellular scales. However

deep-tissue super-resolution imaging remains constrained by background interference

which leads to limited depth penetration and compromised imaging fidelity. To overcome these challenges

we propose a novel imaging system

confocal² spinning-disk image scanning microscopy (C

SD-ISM). It integrates a spinning-disk (SD) confocal microscope

which physically eliminates out-of-focus signals

forming the first confocal level. A digital

micromirror device (DMD) is employed for sparse multifocal illumination

combined with a dynamic pinhole array pixel reassignment (DPA-PR) algorithm for ISM super-resolution reconstruction

forming the second confocal level. The dual confocal configuration enhances system resolution

while effectively mitigating scattering background interference. Compared to computational out-of-focus signal removal

SD preserves the original intensity distribution as the penetration depth increases

achieving an imaging depth of up to 180 μm. Additionally

the DPA-PR algorithm effectively corrects Stokes shifts

optical aberrations

and other non-ideal conditions

achieving a lateral resolution of 144 nm and an axial resolution of 351 nm

and a linear correlation of up to 92% between the original confocal and the reconstructed image

thereby enabling high-fidelity super-resolution imaging. Moreover

the system’s programmable illumination via the DMD allows for seamless realization with structured illumination microscopy modality

offering excellent scalability and ease of use. Altogether

these capabilities make the C

SD-ISM system a versatile tool

advancing cellular imaging and tissue-scale exploration for modern bioimaging needs.

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Keywords

references

Pawley, J. B. Handbook of Biological Confocal Microscopy 3rd edn (Springer, New York, 2006).

Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19 , 780–782 (1994)..

Lukinavičius, G. et al. Stimulated emission depletion microscopy. Nat. Rev. Methods Prim. 4 , 56 (2024)..

Booth, M. et al. Aberrations and adaptive optics in super-resolution microscopy. Microscopy 64 , 251–261 (201 5)..

Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198 , 82–87 (2000)..

Wu, Y. C. & Shroff, H. Faster, sharper, and deeper: structured illumination microscopy for biological imaging. Nat. Methods 15 , 1011–1019 (2018)..

Cao, R. J. et al. Open-3DSIM: an open-source three-dimensional structured illumination microscopy reconstruction platform. Nat. Methods 20 , 1183–1186 (2023)..

Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Prim. 1 , 39 (2021)..

Sheppard, C. J. R. Super-resolution in confocal imaging. Optik 80 , 53–54 (1988)..

Müller, C. B. & Enderlein, J. Image scanning microscopy. Phys. Rev. Lett. 104 , 198101 (2010)..

York, A. G. et al. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat. Methods 9 , 749–754 (2012)..

De Luca, G. M. R. et al. Re-scan confocal microscopy: scanning twice for better resolution. Biomed. Opt. Express 4 , 2644–2656 (2013)..

Roth, S. et al. Optical photon reassignment microscopy (OPRA). Optical Nanoscopy 2 , 5 (2013)..

Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proc. Natl. Acad. Sci. USA 110 , 21000–21005 (2013)..

York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods 10 , 1122–1126 (2013)..

Azuma, T. & Kei, T. Super-resolution spinning-disk confocal microscopy using optical photon reassignment. Opt. Express 23 , 15003–15011 (2015)..

Castello, M. et al. A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM. Nat. Methods 16 , 175–178 (2019)..

Mahecic, D. et al. Homogeneous multifocal excitation for high-throughput super-resolution imaging. Nat. Methods 17 , 726–733 (2020)..

Ren, W. et al. Expanding super-resolution imaging versatility in organisms with multi-confocal image scanning microscopy. Natl Sci. Rev. 11 , nwae303 (2024)..

Xiao, G. Q., Corle, T. R. & Kino, G. S. Real‐time confocal scanning optical microscope. Appl. Phys. Lett. 53 , 716–718 (1988)..

Petráň, M. et al. Tandem-scanning reflected-light microscope. J. Optical Soc. Am. 58 , 661–664 (1968)..

Li, M. Q. et al. Structured illumination microscopy using digital micro-mirror device and coherent light source. Appl. Phys. Lett. 116 , 233702 (2020)..

Halpern, A. R. et al. Versatile, do-it-yourself, low-cost spinning disk confocal microscope. Biomed. Opt. Express 13 , 1102–1120 (2022)..

Tanaami, T. et al. High-speed 1-frame/ms scanning confocal microscope with a microlens and Nipkow disks. Appl. Opt. 41 , 4704–4708 (2002)..

Sheppard, C. J. R., Mehta, S. B. & Heintzmann, R. Superresolution by image scanning microscopy using pixel reassignment. Opt. Lett. 38 , 2889–2892 (2013)..

Castello, M. et al. Image scanning microscopy with a quadrant detector. Opt. Lett. 40 , 5355–5358 (2015)..

Guizar-Sicairos, M., Thurman, S. T. & Fienup, J. R. Efficient Subpixel image registration algorithms. Opt. Lett. 33 , 156–158 (2008)..

Koho, S. et al. Fourier ring correlation simplifies image restoration in fluorescence microscopy. Nat. Commun. 10 , 3103 (2019)..

Culley, S. et al. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat. Methods 15 , 263–266 (2018)..

Sage, D. et al. DeconvolutionLab2: an open-source software for deconvolution microscopy. Methods 115 , 28–41 (2017)..

Sternberg. Biomedical image processing. Computer 16 , 22–34 (1983)..

Hüpfel, M. et al. Wavelet-based background and noise subtraction for fluorescence microscopy images. Biomed. Opt. Express 12 , 969–980 (2021)..

Cao, R. J. et al. Dark-based optical sectioning assists background removal in fluorescence microscopy. Nat. Methods 22 , 1299–1310 (2025)..

Jacques, S. L. Optical properties of biological tissues: a review. Phys. Med. Biol. 58 , R37 (2013)..

Wen, G. et al. High-fidelity structured illumination microscopy by point-spread-function engineering. Light Sci. Appl. 10 , 70 (2021)..

Guo, Y. T. et al. Visualizing intracellular organelle and cytoskeletal interactions at nanoscale resolution on millisecond timescales. Cell 175 , 1430–1442.e17 (2018)..

Li, Y. N. et al. High-speed autopolarization synchronization modulation three-dimensional structured illumination microscopy. Adv. Photonics Nexus 3 , 016001 (2023)..

Zhang, M. et al. Deep learning enhanced light sheet fluorescence micr oscopy for in vivo 4D imaging of zebrafish heart beating. Light Sci. Appl. 14 , 92 (2025)..

Booth, M. J. Adaptive optical microscopy: the ongoing quest for a perfect image. Light Sci. Appl. 3 , e165 (2014)..

Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248 , 73–76 (1990)..

Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13 , 1468–1476 (2005)..

Liu, S. et al. Compressive confocal microscopy imaging at the single-photon level with ultra-low sampling ratios. Commun. Eng. 3 , 88 (2024)..

Ren, W. et al. Visualization of cristae and mtDNA interactions via STED nanoscopy using a low saturation power probe. Light Sci. Appl. 13 , 116 (2024)..

Wang, B. J. et al. Multicomposite super-resolution microscopy: enhanced Airyscan resolution with radial fluctuation and sample expansions. J. Biophotonics 13 , e2419 (2020)..

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High-fidelity tissue super-resolution imaging achieved with confocal2 spinning-disk image scanning microscopy

DOI：10.1038/s41377-025-01930-x

摘要

Abstract

关键词

Keywords

references

High-fidelity tissue super-resolution imaging achieved with confocal² spinning-disk image scanning microscopy