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Vibrational fiber photometry: label-free and reporter-free minimally invasive Raman spectroscopy deep in the mouse brain

Nature Methods volume 22, pages 371–379 (2025)Cite this article

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Abstract

Optical approaches to monitor neural activity are transforming neuroscience, owing to a fast-evolving palette of genetically encoded molecular reporters. However, the field still requires robust and label-free technologies to monitor the multifaceted biomolecular changes accompanying brain development, aging or disease. Here, we have developed vibrational fiber photometry as a low-invasive method for label-free monitoring of the biomolecular content of arbitrarily deep regions of the mouse brain in vivo through spontaneous Raman spectroscopy. Using a tapered fiber probe as thin as 1 µm at its tip, we elucidate the cytoarchitecture of the mouse brain, monitor molecular alterations caused by traumatic brain injury, as well as detect markers of brain metastasis with high accuracy. We view our approach, which introduces a deep learning algorithm to suppress probe background, as a promising complement to the existing palette of tools for the optical interrogation of neural function, with application to fundamental and preclinical investigations of the brain and other organs.

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Fig. 1: Deep brain vibrational photometry with a thin tapered fiber.
Fig. 2: Vibrational photometry for the spectral characterization of melanoma brain metastasis.
Fig. 3: Ratiometric diagnostics.
Fig. 4: Vibrational photometry for the spectral characterization of traumatic brain injury.
Fig. 5: Technological perspectives.

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Data availability

The data underlying the conclusions of this manuscript are available from the corresponding authors and the following link: https://doi.org/10.5281/zenodo.14016545 (ref. 70), under CC BY-NC-SA 4.0.

Code availability

The code underlying the conclusions of this manuscript is available from the corresponding authors and at the following link: https://doi.org/10.5281/zenodo.14016545 (ref. 70), under CC BY-NC-SA 4.0.

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Acknowledgements

This work is dedicated to the memory of Prof. Marco Grande and to his legacy of scientific excellence and human kindness. M.M.-M, E.C., T.J.P., A.B., F.T., L.C., M.D.V., F.D.A., L.M.P., M.V. and F. Pisanello acknowledge funding from the European Union’s Horizon 2020 FET-Open Programme project NANOBRIGHT under grant agreement 828972. F. Pisano, M.B., M.P., L.S., M.D.V. and F. Pisanello acknowledge funding from the European Union’s Horizon 2020 Research and Innovation Programme project DEEPER under grant agreement 101016787. This research received support from the European Community under the program Horizon 2020 project ProID under grant agreement 964363. F. Pisano acknowledges funding from PARD 2024 from the University of Padua. M.M.-M. acknowledges funding from a postdoctoral grant (2023) from the Scientific Foundation of the Spanish Association Against Cancer. F.G. acknowledges funding from Associazione Italiana per la Ricerca sul Cancro – AIRC IG 2021 ID 25656. L.C., M.D.V. and F. Pisanello acknowledge funding from the Project 'RAISE (Robotics and AI for Socio-economic Empowerment)' (code ECS00000035) funded by European Union – NextGenerationEU PNRR MUR – M4C2 – Investimento 1.5 – Avviso 'Ecosistemi dell’Innovazione' CUP J33C22001220001. A.B. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement (101106602).

Author information

Author notes

  1. Deceased: Marco Grande.

  2. These authors jointly supervised this work: Francesco Gentile, Francesco De Angelis, Massimo De Vittorio, Liset Menendez de la Prida, Manuel Valiente, Ferruccio Pisanello.

Authors and Affiliations

  1. Istituto Italiano di Tecnologia, Center for Biomolecular Nanotechnologies, Arnesano, Italy

    Filippo Pisano, Maria Samuela Andriani, Mohammadrahim Kazemzadeh, Antonio Balena, Liam Collard, Marco Bianco, Massimo De Vittorio & Ferruccio Pisanello

  2. Department of Physics and Astronomy, University of Padova, Padova, Italy

    Filippo Pisano

  3. Brain Metastasis group, CNIO, Madrid, Spain

    Mariam Masmudi-Martín, Patricia Baena & Manuel Valiente

  4. Dipartimento di Ingegneria dell’Innovazione, Università del Salento, Lecce, Italy

    Maria Samuela Andriani & Massimo De Vittorio

  5. Instituto Cajal, CSIC, Madrid, Spain

    Elena Cid, Teresa Jurado Parras & Liset Menendez de la Prida

  6. Optogenix srl, Arnesano, Italy

    Marco Pisanello & Leonardo Sileo

  7. Laboratoire Kastler Brossel, Sorbonne University, CNRS, ENS-PSL University, Collège de France, Paris, France

    Antonio Balena

  8. RAISE Ecosystem, Genoa, Italy

    Liam Collard, Massimo De Vittorio & Ferruccio Pisanello

  9. Istituto Italiano di Tecnologia, Center for Convergent Technologies, Genoa, Italy

    Francesco Tantussi & Francesco De Angelis

  10. Dipartimento di Ingegneria Elettrica e dell’Informazione, Bari, Italy

    Marco Grande

  11. Nanotechnology Research Center, Department of Experimental and Clinical Medicine, University of ‘Magna Graecia’ of Catanzaro, Catanzaro, Italy

    Francesco Gentile

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  1. Filippo Pisano
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  2. Mariam Masmudi-Martín
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Contributions

F. Pisano and M.M.M. are co-first authors. M.S.A., E.C. M.K. and M.P. contributed equally and are listed in alphabetical order. F.G., F.D.A., M.D.V., L.M.P., M.V. and F. Pisanello jointly supervised the work and are co-last authors on this work. F. Pisano, M.P., M.G., A.B., F.T., F.D.A., M.D.V. and F. Pisanello developed the bench-top Raman system. M.P., M.S.A., F.T., F. Pisano and F. Pisanello developed the portable Raman system. M.M.-M., M.V., T.J.P. and L.M.P. tested the portable system. L.S. developed the tapered optical fiber for the Raman application. M.S.A., M.P. and F. Pisano performed the ex vivo experiments. M.M.-M., P.B. and M.V. developed the melanoma brain metastasis model and performed the in vivo experiments. E.C., T.J.P. and L.M.P. developed the traumatic brain injury model and carried out the immunohistochemistry. T.J.P., E.C., M.S.A. and L.M.P. performed the in vivo traumatic brain injury experiments and collected Raman data. F. Pisano, M.S.A., M.P., M.B., A.B., L.C., F.G. and F. Pisanello developed and performed the data analysis. M.K. developed and performed the deep learning network and analysis. M.B., M.G. and F. Pisano developed and performed the numerical simulations. F. Pisano and A.B. fabricated the micro-structured probes and performed the experiments. F.G. developed and performed the principal component analysis. F. Pisano, F.D.A., M.D.V., L.M.P., M.V. and F. Pisanello conceived the application of vibration fiber photometry in deep brain regions. F. Pisano and F. Pisanello conceptualized the work with contributions from all authors. F. Pisano drafted the manuscript and prepared the figures. All of the authors edited the manuscript.

Corresponding authors

Correspondence to Filippo Pisano or Ferruccio Pisanello.

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Competing interests

L.S., M.D.V. and F. Pisanello are founders and hold private equity in Optogenix, a company that develops, produces and sells technologies to deliver light into the brain. L.S. and M.P. are employed by Optogenix. F. Pisano has been employed by Optogenix. Products of Optogenix have been used in this research. M.V. receives research funds from AstraZeneca. All other authors have no competing interests.

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Nature Methods thanks Hyeon Jeong Lee, Wei Min, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 Histology and reconstruction of fiber tracks.

a, Comparison of histology and PCA prediction for melanoma brain metastasis in ex vivo brain. Histological classification is overlaid with histological images for healthy tissue (green) and tissue with metastatic cells (red) within the collection area of the probe. Similarly, PCA classification in cluster 1, associated with healthy tissue (cyan), and cluster 2, associated with cancer (magenta),in the fingerprint (FP) (filled circles) and high-wavenumber (HW) (empty circles) ranges. b, Images of the fiber track position after in vivo experiments generating the spectra shown as orange dots in the PCA clusters in Fig. 2n. The images are acquired in fixed brain slices. The fiber track, pointed by red arrows, narrowly misses the brain metastasis. The image on the left was acquired on a brightfield fluorescence microscope; the image on the right was obtained by processing the left image with a high-pass filter to highlight the fiber track.

Extended Data Fig. 2 Supplementary data for ratiometric analysis.

a, Example of the raw Raman spectra used for ratiometric analysis acquired in metastatic tissue (magenta) and in a contralateral position in healthy tissue (cyan). b Derivative of Raman spectra for cancer free (cyan) and cancer invade (magenta) regions in the high-wavenumber (HW) range. For display reasons, the plot shows 1 measurement out of 5 for every implant position.

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Pisano, F., Masmudi-Martín, M., Andriani, M.S. et al. Vibrational fiber photometry: label-free and reporter-free minimally invasive Raman spectroscopy deep in the mouse brain. Nat Methods 22, 371–379 (2025). https://doi.org/10.1038/s41592-024-02557-3

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