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Three-photon microscopy: an emerging technique for deep intravital brain imaging

Nature Reviews Neuroscience volume 26, pages 521–537 (2025)Cite this article

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Abstract

Understanding brain function and pathology requires observation of cellular dynamics within intact neural circuits. Although two-photon microscopy revolutionized mammalian in vivo brain imaging, its limitation to upper cortical layers has restricted access to many important brain regions. Three-photon microscopy overcomes this constraint, enabling minimally invasive yet high-resolution visualization of the deep cortical and subcortical structures that are crucial for higher-order brain functions. This emerging technology opens new avenues for investigating fundamental aspects of neuroscience, from circuit dynamics to disease mechanisms. Here, we examine how three-photon microscopy has started to transform our ability to investigate neural circuits, glial biology, and oncological and neuroimmune interactions in previously inaccessible brain regions, primarily in the mouse, but also in other model organisms. We discuss current technical challenges, recent innovations and future applications that promise to bring us greater understanding of the living brain.

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Fig. 1: Principles of three-photon microscopy for intravital imaging.
Fig. 2: Examples of three-photon microscopy in different animal models.
Fig. 3: Future directions for three-photon microscopy.

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Acknowledgements

We thank P. Rupprecht and F. Xia for the valuable feedback and critical reading of this manuscript. This work was supported by the European Molecular Biology Laboratory (J.F.O. and R.P.). R.P. acknowledges support from the European Commission (ERC Consolidator Grant 864027, Brillouin4Life) and the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (projects 2020-225346 and 2024-337799). J.F.O. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement number 945405. M.B. acknowledges funding from the Emmy Noether Program of the German Research Foundation (DFG; Grant BR 6153/1-1) and the Else Kröner Fresenius Stiftung (2024_EKCS.06). V.V. and M.O.B. were supported by the Collaborative Research Center (CRC) 1389 (UNITE Glioblastoma). J.W. received funding from the National Institutes of Health (NS115585, NS121766). V.V. was supported by the DFG (project number VE1373/2-1516). V.V. received financial support from Else Kröner–Fresenius–Stiftung (2020-EKEA.135), the European Center for Neurooncology, the Health + Life Science Alliance Heidelberg Mannheim (together with R.P.), Heidelberg University, the Schwiete-Stiftung, the Wilhelm-Sander-Stiftung, the Stiftung Sibylle Assmus and Research Seed Capital from the Ministry of Science, Research and the Arts Baden Württemberg.

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Authors and Affiliations

  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Robert Prevedel & Júlia Ferrer Ortas

  2. Epigenetics and Neurobiology Unit, European Molecular Biology Laboratory, Monterotondo, Italy

    Robert Prevedel

  3. Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory, Heidelberg, Germany

    Robert Prevedel & Júlia Ferrer Ortas

  4. Interdisciplinary Center for Neurosciences, Heidelberg University, Heidelberg, Germany

    Robert Prevedel

  5. Department of Behavior and Brain Organization, Max Planck Institute for Neurobiology of Behavior, Bonn, Germany

    Jason N. D. Kerr

  6. Allen Institute for Brain Science, Seattle, WA, USA

    Jack Waters

  7. Neuroradiology Department, Division of Immuno-Imaging, University Hospital Heidelberg, Heidelberg, Germany

    Michael O. Breckwoldt

  8. Department of Neurosurgery, Baylor College of Medicine, Houston, TX, USA

    Benjamin Deneen

  9. Center for Cancer Neuroscience, Baylor College of Medicine, Houston, TX, USA

    Benjamin Deneen

  10. Department of Neurology, Stanford University, Stanford, CA, USA

    Michelle Monje

  11. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Michelle Monje

  12. Neurology Clinic, University Hospital Heidelberg, Heidelberg, Germany

    Stella J. Soyka & Varun Venkataramani

  13. European Center for Neurooncology, University Hospital Heidelberg, Heidelberg, Germany

    Stella J. Soyka & Varun Venkataramani

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Contributions

R.P., J.F.O., J.W., S.J.S. and V.V. researched data for the article. R.P., J.N.D.K., J.W., M.M. and V.V. contributed substantially to the discussion of the content. R.P., J.F.O., J.N.D.K., J.W., M.O.B., B.D., S.J.S. and V.V. wrote the article and reviewed and/or edited the manuscript before submission. R.P. and V.V. coordinated and led the writing of the article.

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Correspondence to Robert Prevedel or Varun Venkataramani.

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Nature Reviews Neuroscience thanks Ethan Hughes; Cristina Rodríguez, who co-reviewed with Malika Datta; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Glossary

Absorption cross section

A measure of the probability that a photon will be absorbed to excite a (fluorescence) molecule, determining the efficiency of fluorescence signal generation.

Adaptive optics

A technology that corrects for wavefront distortions caused by the sample or imaging system, thereby improving image resolution and signal in deep and/or scattering biological tissues.

Attenuation coefficient

A parameter describing the reduction in light intensity as it propagates through a medium, owing to scattering and absorption.

Confocal microscopy

An imaging technique that uses a spatial pinhole to block out-of-focus light, thereby producing high-contrast optical sections of thick specimens.

Dual-plane imaging

A microscopy method that simultaneously captures images from two different focal planes, enabling faster acquisition of three-dimensional data.

Guidestars

Artificial or natural reference points emitting (fluorescence) signals, used in adaptive optics systems to measure and correct wavefront distortions for improved resolution and image quality.

Multiphoton microscopy

An advanced imaging method wherein two or more low-energy photons are absorbed simultaneously to excite a fluorophore, enabling deep tissue imaging with high resolution and minimal photodamage.

Optical sectioning

The ability of an imaging system to isolate thin slices of a sample along the axial direction, improving resolution and contrast in three-dimensional imaging.

Optical wavefront shaping

The manipulation of light wavefronts using devices such as spatial light modulators to control and correct for aberrations introduced by complex, highly scattering media.

Remote focusing

A technique to adjust the focal plane of a microscope rapidly and precisely by manipulating the optical path without moving the primary objective lens.

Third-order harmonic signal

A nonlinear optical signal generated when three photons combine to produce a single photon with triple the energy (or one-third the wavelength), enabling visualization of intrinsic sample structures without fluorescent labels.

Volumetric imaging

A method for capturing 3D data sets by acquiring images across multiple depths, enabling detailed reconstruction of structures in three dimensions.

Wavefront distortions

Aberrations in the light wavefront caused by refractive index variations in a sample, degrading image quality in microscopy.

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Prevedel, R., Ferrer Ortas, J., Kerr, J.N.D. et al. Three-photon microscopy: an emerging technique for deep intravital brain imaging. Nat. Rev. Neurosci. 26, 521–537 (2025). https://doi.org/10.1038/s41583-025-00937-y

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