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Fabrication of nonplanar tapered fibers to integrate optical and electrical signals for neural interfaces in vivo

Nature Protocols volume 20, pages 1768–1809 (2025)Cite this article

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Abstract

Implantable multifunctional probes have transformed neuroscience research, offering access to multifaceted brain activity that was previously unattainable. Typically, simultaneous access to both optical and electrical signals requires separate probes, while their integration into a single device can result in the emergence of photogenerated electrical artifacts, affecting the quality of high-frequency neural recordings. Among the nontrivial strategies aimed at the realization of an implantable multifunctional interface, the integration of optical and electrical capabilities on a single, minimally invasive, tapered optical fiber probe has been recently demonstrated using fibertrodes. Fibertrodes require the application of a set of planar microfabrication techniques to a nonplanar system with low and nonconstant curvature radius. Here we develop a process based on multiple conformal depositions, nonplanar two-photon lithography and chemical wet etching steps to obtain metallic patterns on the highly curved surface of the fiber taper. We detail the manufacturing, encapsulation and back end of the fibertrodes. The design of the probe can be adapted for different experimental requirements. Using the optical setup design, it is possible to perform angle selective light coupling with the fibertrodes and their implantation and use in vivo. The fabrication of fibertrodes is estimated to require 5–9 d. Nonetheless, due to the high scalability of a large part of the protocol, the manufacture of multiple fibertrodes simultaneously substantially reduces the required time for each probe. The procedure is suitable for users with expertise in microfabrication of electronics and neural recordings.

Key points

  • The procedure covers microfabrication approaches including the conformal deposition of metallic and dielectric thin films, nonplanar two-photon lithography, wet etching and focused ion beam milling/deposition.

  • The integration of electrical and optical signals within a single device enables the optical control of neural activity and the recording of simultaneous electrophysiological readout in mice. Alternative approaches include the use of flexible electronics, micro light-emitting diodes, multifunctional polymeric optical fibers and silica-based arrays.

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Fig. 1: Flowchart for the fabrication and usage of fibertrodes.
Fig. 2: TPP-based process to fabricate metallic patterns on TFs.
Fig. 3: Design principles for the fibertrodes.
Fig. 4: Encapsulation with Parylene-C.
Fig. 5: Monte Carlo simulation of light emitted from the apertures and design principles.
Fig. 6: Preparation of the TFs.
Fig. 7: Fabrication of dielectric apertures.
Fig. 8: Setup and software for TPP of electrode patterns.
Fig. 9: Back-end procedure to connect the patterned TF with the 3D-PCB.
Fig. 10: Depth-resolved optogenetic setup.
Fig. 11: Implantation procedures for fibertrode devices.
Fig. 12: Anticipated results for the optical and the electrophysiological interface.

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Data availability

The authors declare that the main data discussed in this protocol are available in the supporting primary research paper (https://doi.org/10.1038/s41563-022-01272-8) and are available in the Zenodo repository via https://doi.org/10.5281/zenodo.6477861 (ref. 59).

Code availability

The scripts related to the Monte Carlo simulations have been published in ref. 21 and are available in the Zenodo repository via https://doi.org/10.5281/zenodo.6477861 (ref. 59). The driving software of the custom TPP setup is available at Zenodo via https://doi.org/10.5281/zenodo.12773241 (ref. 60), along with a 3D computer-aided design (CAD) model and a detailed list of all the elements of the system at Zenodo via https://doi.org/10.5281/zenodo.6536237 (ref. 28).

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Acknowledgements

A.B., M.B., F.Pisano B.S. and F. Pisanello acknowledge funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (#677683). M.P. and M.D.V. acknowledge funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (#692943). M.B., C.M., M.D.V. and F. Pisanello acknowledge funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (#966674). M.B., F.Pisano, M.D.V. and F.Pisanello. acknowledge that this project has received funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement no. 101016787. M.D.V. is funded by the US National Institutes of Health (U01NS094190). M.P., F.Pisanello and M.D.V. are funded by the US National Institutes of Health (1UF1NS108177-01). A.B. acknowledges funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (no. 101106602). A.B., F.Pisanello and M.D.V. also acknowledge funding from the European Union’s Horizon 2020 research and innovation program under a grant agreement (no. 828972). C.M., B.S., F.Pisanello and M.D.V. acknowledge funding from 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. F. Pisano acknowledges funding from PARD 2024 from the University of Padua.

Author information

Author notes

  1. These authors jointly supervised this work: Bernardo L. Sabatini, Massimo De Vittorio, Ferruccio Pisanello.

Authors and Affiliations

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

    Antonio Balena, Marco Bianco, Maria Samuela Andriani, Cinzia Montinaro, Barbara Spagnolo, Filippo Pisano, Massimo De Vittorio & Ferruccio Pisanello

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

    Antonio Balena

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

    Maria Samuela Andriani & Massimo De Vittorio

  4. OptogeniX S.r.l., Arnesano, Italy

    Marco Pisanello

  5. Dipartimento di Fisica e Astronomia ‘Galileo Galilei’, Università di Padova, Padova, Italy

    Filippo Pisano

  6. Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA

    Bernardo L. Sabatini

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Contributions

A.B., M.B. and M.P. designed and implemented the fabrication protocol. M.P. and F.Pisano designed the optical setup for depth-resolved optogenetic stimulation. C.M. and B.S. conceived the in vivo probe implantation routine. A.B. wrote the paper with extensive contributions from M.S.A. and M.B. on the fabrication part, M.P and F.Pisano on the optical setup design part and C.M. and B.S. on the in vivo implantation part. F. Pisanello, M.D.V. and B.L.S. jointly supervised the work. All the authors discussed the results and commented on the manuscript at all stages. All the authors contributed extensively to the work.

Corresponding authors

Correspondence to Antonio Balena, Massimo De Vittorio or Ferruccio Pisanello.

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Competing interests

F.Pisanello, M.D.V. and B.L.S. are founders and hold private equity in Optogenix, a company that develops, produces and sells technologies to deliver light into the brain. Tapered fibers commercially available from Optogenix were used as tools in the research. The other authors do not declare any competing interests.

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Nature Protocols thanks Angelique Park, Seongjun Park and the other, anonymous, reviewer(s) for the peer review of this work.

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Key reference

Spagnolo, B. et al. Tapered fibertrodes for optoelectrical neural interfacing in small brain volumes with reduced artefacts. Nat. Mater. https://doi.org/10.1038/s41563-022-01272-8 (2022)

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Balena, A., Bianco, M., Andriani, M.S. et al. Fabrication of nonplanar tapered fibers to integrate optical and electrical signals for neural interfaces in vivo. Nat Protoc 20, 1768–1809 (2025). https://doi.org/10.1038/s41596-024-01105-9

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