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Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping

Nature Biotechnology volume 40, pages 1663–1671 (2022)Cite this article

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Abstract

High-resolution optical imaging deep in tissues is challenging because of optical aberrations and scattering of light caused by the complex structure of living matter. Here we present an adaptive optics three-photon microscope based on analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS). ALPHA-FSS accurately measures and effectively compensates for both aberrations and scattering induced by specimens and recovers subcellular resolution at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 µm below the pia, enabling near-non-invasive high-resolution microscopy in cortex. Functional calcium imaging with high sensitivity and high-precision laser-mediated microsurgery through the intact skull were also demonstrated. Moreover, we achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below the pia within the intact brain.

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Fig. 1: Conjugate α-FSS–3PM system and correction of aberrations induced by a 100-µm-thick intact mouse skull.
Fig. 2: Conjugate α-FSS–3PM enables in vivo cortical imaging with high resolution over large volumes through the intact skull.
Fig. 3: Conjugate α-FSS–3PM improves in vivo functional calcium imaging of neuronal activity through the intact skull.
Fig. 4: Conjugate α-FSS–3PM enables precise laser micro-lesion and in vivo high-resolution imaging of microglia in aged AD brain through the intact skull.
Fig. 5: Pupil α-FSS–3PM enables in vivo imaging of deep-cortical and hippocampal neurons at synaptic resolution through an open skull window.

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Data availability

The authors declare that the main data supporting the findings of this study are available within the paper, its extended data and Supplementary Information files. The source data files for all data presented within the figures can be found at https://github.com/QuLabHKUST/QuLabAO.

Code availability

The custom codes for image processing are available online at https://github.com/QuLabHKUST/QuLabAO.

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Acknowledgements

This work was supported by the Hong Kong Research Grants Council through grants 16103215, 16148816, 16102518, 16102920, T13-607/12R, T13-605/18W, C6002-17GF, C6001-19E and N_HKUST603/19 (to J.Y.Q.), the Innovation and Technology Commission (ITCPD/17-9 to N.Y.I.), the Area of Excellence Scheme of the University Grants Committee (AoE/M-604/16 to N.Y.I. and J.Y.Q.), the National Key R&D Program of China (2018YFE0203600 to N.Y.I.) and the Guangdong Provincial Fund for Basic and Applied Basic Research (2019B1515130004 to N.Y.I.). We thank J. He, M. M. Hossian and M. Chen from City University of Hong Kong for providing the CCK-GCaMP6s transgenic mice and preparing the open skull window.

Author information

Author notes

  1. These authors contributed equally: Zhongya Qin, Zhentao She.

Authors and Affiliations

  1. Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

    Zhongya Qin, Zhentao She, Congping Chen, Wanjie Wu & Jianan Y. Qu

  2. Division of Life Science, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

    Jackie K. Y. Lau & Nancy Y. Ip

  3. State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

    Jackie K. Y. Lau, Nancy Y. Ip & Jianan Y. Qu

  4. Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China

    Jackie K. Y. Lau, Nancy Y. Ip & Jianan Y. Qu

  5. Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China

    Jackie K. Y. Lau & Nancy Y. Ip

  6. Guangdong Provincial Key Laboratory of Brain Science, Disease and Drug Development, HKUST Shenzhen Research Institute, Shenzhen-Hong Kong Institute of Brain Science, Shenzhen, China

    Nancy Y. Ip

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Contributions

Z.Q. and J.Y.Q. conceived of the research idea. Z.Q. built the AO 3P imaging system and created the control software. Z.Q., Z.S. and C.C. designed and conducted the experiments and data analysis. Z.S. carried out the surgery, with the assistance of C.C., Z.Q., W.W. and J.L. N.Y.I. and J.Y.Q. supervised the project. C.C. and Z.Q. took the lead in writing the manuscript, with input from all other authors.

Corresponding author

Correspondence to Jianan Y. Qu.

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Competing interests

Z.Q. and J.Y.Q. have submitted a patent application on part of the described work. The remaining authors declare no competing interests.

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Nature Biotechnology thanks Xi Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figs. 1–23 and Supplementary Table 1

Reporting Summary

Supplementary Video 1

Video 1: Conjugate AO with remote focusing enables effective improvement of imaging resolution over large imaging depths ranged from 100 µm to 500 µm, with a single corrective wavefront at 300 µm

Supplementary Video 2

Video 2: Near-simultaneous multi-plane calcium imaging of neuronal and dendritic activities from different cortical layers through the intact skull

Supplementary Video 3

Video 3: Time-lapse imaging at multiple depths revealed that the highly-localized lesion activated only a few adjacent microglia (within a distance of 50 µm)

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Qin, Z., She, Z., Chen, C. et al. Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping. Nat Biotechnol 40, 1663–1671 (2022). https://doi.org/10.1038/s41587-022-01343-w

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