Abstract
Two-photon microscopy, combined with the appropriate optical labelling, enables the measurement and tracking of submicrometer structures within brain cells, as well as the spatiotemporal mapping of spikes in individual neurons and of neurotransmitter release in individual synapses. Yet, the spatial resolution of two-photon microscopy rapidly degrades as imaging is attempted at depths of more than a few scattering lengths into tissue, i.e., below the superficial layers that constitute the top 300–400 µm of the neocortex. To obviate this limitation, we shape the focal volume, generated by the excitation beam, by modulating the incident wavefront via guidestar-assisted adaptive optics. Here, we describe the construction, calibration and operation of a two-photon microscope that incorporates adaptive optics to restore diffraction-limited resolution at depths close to 900 µm in the mouse cortex. Our setup detects a guidestar formed by the excitation of a red-shifted dye in blood serum, used to directly measure the wavefront. We incorporate predominantly commercially available optical, optomechanical, mechanical and electronic components, and supply computer-aided design models of other customized components. The resulting adaptive optics two-photon microscope is modular and allows for expanded imaging and optical excitation capabilities. We demonstrate our methodology in the mouse neocortex by imaging the morphology of somatostatin-expressing neurons that lie 700 µm beneath the pia, calcium dynamics of layer 5b projection neurons and thalamocortical glutamate transmission to L4 neurons. The protocol requires ~30 d to complete and is suitable for users with graduate-level expertise in optics.
Key points
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The resolution of two-photon microscopy degrades when imaging deep into tissue, limiting its effectiveness beyond the superficial layer of neocortex. To overcome this limitation, the authors describe the construction and operation of a two-photon microscope that incorporates adaptive optics to restore diffraction-limited resolution for imaging structure and function in deep layers.
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The red-shifted dye cyanine 5.5-conjugated 2,000 kDa dextran is readily delivered to the blood serum and serves as a guidestar for adaptive optics.
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Data availability
The data used for generating the time series in Figs. 17 and 18 can be accessed via https://dandiarchive.org/dandiset/000454/0.230302.2331.
Code availability
The MATLAB scripts for calibrating and operating the AO-TPLSM are available in the Supplementary Software.
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Acknowledgements
We thank S. Adams for assistance with Cy5.5–dextran synthesis and B. Friedman for assistance with animal preparations. This work was funded by the National Science Foundation, grant PHY no. 1532264; the National Institutes of Health, grants U24 EB028942, R35 NS097265 and U19 NS107466; and Early and Advanced Postdoc Mobility fellowships (P2SKP3_164948 and P300PA_177804) from the Swiss National Science Foundation.
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Contributions
D.K. and R.L. guided this project and R.L. and P.Y. designed the microscope, which is based on a prior design by R.L. P.Y. carried out fabrication and testing of the hardware, T.B., R.L. and M.T. developed the method to synthesize the Cy5.5–dextran, R.L. and P.Y. performed all in vivo measurements, and D.K. and P.Y. wrote the manuscript. D.K. attended to the plethora of university rules and forms that govern research compliance, export control and environmental health and safety, including the ethical use of animals as well as the use of chemicals, hazardous substances, controlled substance, lasers and viruses.
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Nature Protocols thanks Valentina Emiliani, Karen Hampson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Liu, R. et al. Nat. Methods 16, 615–618 (2019): https://doi.org/10.1038/s41592-019-0434-7
Supplementary information
Supplementary Data 1
Assembly drawings.
Supplementary Data 2
Part drawings.
Supplementary Data 3
ZEMAX file.
Supplementary Table 1
Comprehensive parts list including current prices and vendor links.
Supplementary Code 1
MATLAB scripts.
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Yao, P., Liu, R., Broggini, T. et al. Construction and use of an adaptive optics two-photon microscope with direct wavefront sensing. Nat Protoc 18, 3732–3766 (2023). https://doi.org/10.1038/s41596-023-00893-w
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DOI: https://doi.org/10.1038/s41596-023-00893-w
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