Second-level high-speed 3D isotropic imaging of whole mouse brain using deep-learning spinning-disk light-sheet microscopy

Fang Zhao; Junyu Ping; Xingyu Chen; Yuyi Wang; Zhaofei Wang; Jingtan Zhu; Chaoliang Ye; Yuan Wang; Man Jiang; Dan Zhu; Fenghe Zhong; Yuxuan Zhao; Peng Fei

doi:10.1186/s43074-025-00200-8

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Fang Zhao, Junyu Ping, Xingyu Chen, Yuyi Wang, Zhaofei Wang, Jingtan Zhu, Chaoliang Ye, Yuan Wang, Man Jiang, Dan Zhu, Fenghe Zhong, Yuxuan Zhao, Peng Fei. Second-level high-speed 3D isotropic imaging of whole mouse brain using deep-learning spinning-disk light-sheet microscopy[J]. PhotoniX. doi: 10.1186/s43074-025-00200-8

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Second-level high-speed 3D isotropic imaging of whole mouse brain using deep-learning spinning-disk light-sheet microscopy

doi: 10.1186/s43074-025-00200-8

1.
School of Optical and Electronic Information-Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, China
2.
Advanced Biomedical Imaging Facility, Huazhong University of Science and Technology, Wuhan, 430074, Hubei, China
3.
Britton Chance Center and MoE Key Laboratory for Biomedical Photonics, Wuhan National Laboratory for Optoelectronic, Huazhong University of Science and Technology, Wuhan, 430074, China
4.
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China

Funds: This work was supported by the funding from National Natural Science Foundation of China (T2225014, 82270238, 21927802, 22404065, 62405099). National Key Research and Development Program of China (2022YFC3401102, 2023ZD0519900). China Postdoctoral Science Foundation (2024T170296, 2023M741258, 2024M750994). Postdoctor Project of Hubei Province under Grant Number (2024HBBHCXA015).

Received Date: 2025-04-12
Accepted Date: 2025-09-20
Rev Recd Date: 2025-09-11

Available Online: 2025-10-15

Abstract

Abstract

Axially-swept light-sheet microscopy (ASLM) has emerged as a distinguished tool for 3D imaging owing to its excellent spatial resolution. However, the acquisition time is significantly elongated due to the extra time consumed in axial scanning. Meanwhile, the spatial information provided in a single scan is fundamentally limited by the compromise between field-of-view and resolution. The overall inadequate optical throughput of current ASLM techniques impedes their widespread application in acquiring large samples. Here we demonstrate a spinning-disk-based ASLM (SDLM) approach that enables wide field-of-view (15 × confocal range of the gaussian beam), isotropic 3D imaging of large organisms at 100 Hz full camera frame rate. In addition to the new optical design, we combine a recurrent neural network image restoration model to further improve the resolution of raw images. We demonstrate seconds scale stitching-free 3D imaging of the entire mouse brain (~ 9*8*5 mm size) at isotropic single-cell resolution (1.5 µm voxel). With the high-quality data readily obtained by our approach, we also demonstrate the visualization of long projecting neurons and two genotypes of whole mouse brain cell profiling across the 3D space. Further transformation into in vivo research would broaden the application of SDLM.
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References

[1]	Jonkman J, Brown CM, Wright GD, Anderson KI, North AJ. Tutorial: guidance for quantitative confocal microscopy. Nat Protoc. 2020;15:1585–611. https://doi.org/10.1038/s41596-020-0313-9.
[2]	Qian J, Wang C, Wu H, Chen Q, Zuo C. Ensemble Deep Learning-enabled single-shot composite Structured Illumination Microscopy (eDL-cSIM). PhotoniX. 2025;6. https://doi.org/10.1186/s43074-025-00171-w.
[3]	Zhang C, Yu B, Lin F, Samanta S, Yu H, Zhang W, et al. Deep tissue super-resolution imaging with adaptive optical two-photon multifocal structured illumination microscopy. PhotoniX. 2023;4. https://doi.org/10.1186/s43074-023-00115-2.
[4]	Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science. 2004;305:1007–9. https://doi.org/10.1126/science.1100035.
[5]	Dodt HU, Leischner U, Schierloh A, Jahrling N, Mauch CP, Deininger K, et al. Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods. 2007;4:331–6.
[6]	Vladimirov N, Voigt FF, Naert T, Araujo GR, Cai R, Reuss AM, et al. Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples. Nat Commun. 2024;15:2679. https://doi.org/10.1038/s41467-024-46770-2.
[7]	Ahrens MB, Orger MB, Robson DN, Li JM, Keller PJ. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods. 2013;10:413–20. https://doi.org/10.1038/nmeth.2434.
[8]	Erturk A, Becker K, Jahrling N, Mauch CP, Hojer C, Egen J, et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc. 2012;17:1983–95.
[9]	Pan C, Cai R, Quacquarelli FP, Ghasemigharagoz A, Lourbopoulos A, Matryba P, et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods. 2016;13:859–67. https://doi.org/10.1038/nmeth.3964.
[10]	Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc. 2015;10:1709–27. https://doi.org/10.1038/nprot.2015.085.
[11]	Tomer R, Ye L, Hsueh B, Deisseroth K. Advanced clarity for rapid and high-resolution imaging of intact tissues. Nat Protoc. 2014;9:1682–97.
[12]	Glaser AK. A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues. Nat Methods. 2022;19:613.
[13]	Yang B, Lange M, Millett-Sikking A, Zhao X, Bragantini J, VijayKumar S, et al. Daxi—high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy. Nat Methods. 2022;19:461–9. https://doi.org/10.1038/s41592-022-01417-2.
[14]	Zhao F, Yang Y, Li Y, Jiang H, Xie X, Yu T, Wang X, Liu Q, Zhang H, Jia H, et al. Efficient and cost‐effective 3D cellular imaging by sub‐voxel‐resolving light‐sheet add‐on microscopy. J Biophotonics. 2020;13. https://doi.org/10.1002/jbio.201960243.
[15]	Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods. 2011;8:417–23. https://doi.org/10.1038/nmeth.1586.
[16]	Gao L, Shao L, Higgins C, Poulton J, Peifer M, Davidson M, et al. Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell. 2012;151:1370–85.
[17]	Chen BC, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science. 2014;346:1257998. https://doi.org/10.1126/science.1257998.
[18]	Fang C, Yu T, Chu T, Feng W, Zhao F, Wang X, Huang Y, Li Y, Wan P, Mei W, et al. Minutes-timescale 3D isotropic imaging of entire organs at subcellular resolution by content-aware compressed-sensing light-sheet microscopy. Nat Commun. 2021;12:107.
[19]	Vettenburg T, Dalgarno HIC, Nylk J, Coll-Lladó C, Ferrier DEK, Čižmár T, et al. Light-sheet microscopy using an airy beam. Nat Methods. 2014;11:541–4. https://doi.org/10.1038/nmeth.2922.
[20]	Xiong B, Han X, Wu J, Xie H, Dai Q. Improving axial resolution of Bessel beam light-sheet fluorescence microscopy by photobleaching imprinting. Opt Express. 2020;28:9464. https://doi.org/10.1364/OE.388808.
[21]	Dean KM, Chakraborty T, Daetwyler S, Lin J, Garrelts G, M’Saad O, et al. Isotropic imaging across spatial scales with axially swept light-sheet microscopy. Nat Protoc. 2022;17:2025–53. https://doi.org/10.1038/s41596-022-00706-6.
[22]	Dean KM. Deconvolution-free subcellular imaging with axially swept light sheet microscopy. Biophys J. 2015;108:2807–15.
[23]	Ping J, Zhao F, Nie J, Yu T, Zhu D, Liu M, et al. Propagating-path uniformly scanned light sheet excitation microscopy for isotropic volumetric imaging of large specimens. J Biomed Opt. 2019;24:1. https://doi.org/10.1117/1.JBO.24.8.086501.
[24]	Daetwyler S, Mazloom-Farsibaf H, Zhou FY, Segal D, Sapoznik E, Chen B, et al. Imaging of cellular dynamics from a whole organism to subcellular scale with self-driving, multiscale microscopy. Nat Methods. 2025. https://doi.org/10.1038/s41592-025-02598-2.
[25]	Migliori B, Datta MS, Dupre C, Apak MC, Asano S, Gao R, et al. Light sheet theta microscopy for rapid high-resolution imaging of large biological samples. BMC Biol. 2018;16:57. https://doi.org/10.1186/s12915-018-0521-8.
[26]	Voigt FF, Kirschenbaum D, Platonova E, Pagès S, Campbell RAA, Kastli R, et al. The mesoSPIM initiative: open-source light-sheet microscopes for imaging cleared tissue. Nat Methods. 2019;16:1105–8. https://doi.org/10.1038/s41592-019-0554-0.
[27]	Chakraborty T, Driscoll MK, Jeffery E, Murphy MM, Roudot P, Chang BJ, et al. Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution. Nat Methods. 2019;16:1109–13. https://doi.org/10.1038/s41592-019-0615-4.
[28]	Dibaji H, Prince MNH, Yi Y, Zhao H, Chakraborty T. Axial scanning of dual focus to improve light sheet microscopy. Biomed Opt Express. 2022;13:4990. https://doi.org/10.1364/BOE.464292.
[29]	Wang Y, Gong J, Xu N, Yan S, Dong D, Shi K. Large field of view and isotropic light sheet microscopy with aberration-free tunable foci. Laser Photon Rev. 2025;19:2400214. https://doi.org/10.1002/lpor.202400214.
[30]	Jing D, Zhang S, Luo W, Gao X, Men Y, Ma C, et al. Tissue clearing of both hard and soft tissue organs with the PEGASOS method. Cell Res. 2018;28:803–18. https://doi.org/10.1038/s41422-018-0049-z.
[31]	Weigert M, Schmidt U, Boothe T, Müller A, Dibrov A, Jain A, et al. Content-aware image restoration: pushing the limits of fluorescence microscopy. Nat Methods. 2018;15:1090–7. https://doi.org/10.1038/s41592-018-0216-7.
[32]	Zhong Y, Yu Y, Li Y, Yin J, Sun Y, Jiang R, et al. Terahertz photons promote neuron growth and synapse formation through cAMP signaling pathway. PhotoniX. 2025;6. https://doi.org/10.1186/s43074-025-00165-8.
[33]	Wang X, Zeng W, Yang X, Zhang Y, Fang C, Zeng S, et al. Bi-channel image registration and deep-learning segmentation (BIRDS) for efficient, versatile 3D mapping of mouse brain. Elife. 2021;10:e63455. https://doi.org/10.7554/eLife.63455.
[34]	Harris JA, Hirokawa KE, Sorensen SA, Gu H, Mills M, Ng LL, Bohn P, Mortrud M, Ouellette B, Kidney J, et al. Anatomical characterization of cre driver mice for neural circuit mapping and manipulation. Front Neural Circuits. 2014;8. https://doi.org/10.3389/fncir.2014.00076.
[35]	Vong L, Ye C, Yang Z, Choi B, Chua S, Lowell BB. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron. 2011;71:142–54. https://doi.org/10.1016/j.neuron.2011.05.028.
[36]	Xie H, Han X, Xiao G, Xu H, Zhang Y, Zhang G, et al. Multifocal fluorescence video-rate imaging of centimetre-wide arbitrarily shaped brain surfaces at micrometric resolution. Nat Biomed Eng. 2023;8:740–53. https://doi.org/10.1038/s41551-023-01155-6.