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  • Protocol Extension
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Customizable, wireless and implantable neural probe design and fabrication via 3D printing

Nature Protocols volume 18, pages 3–21 (2023)Cite this article

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Abstract

This Protocol Extension describes the low-cost production of rapidly customizable optical neural probes for in vivo optogenetics. We detail the use of a 3D printer to fabricate minimally invasive microscale inorganic light-emitting-diode-based neural probes that can control neural circuit activity in freely behaving animals, thus extending the scope of two previously published protocols describing the fabrication and implementation of optoelectronic devices for studying intact neural systems. The 3D-printing fabrication process does not require extensive training and eliminates the need for expensive materials, specialized cleanroom facilities and time-consuming microfabrication techniques typical of conventional manufacturing processes. As a result, the design of the probes can be quickly optimized, on the basis of experimental need, reducing the cost and turnaround for customization. For example, 3D-printed probes can be customized to target multiple brain regions or scaled up for use in large animal models. This protocol comprises three procedures: (1) probe fabrication, (2) wireless module preparation and (3) implantation for in vivo assays. For experienced researchers, neural probe and wireless module fabrication requires ~2 d, while implantation should take 30–60 min per animal. Time required for behavioral assays will vary depending on the experimental design and should include at least 5 d of animal handling before implantation of the probe, to familiarize each animal to their handler, thus reducing handling stress that may influence the result of the behavioral assays. The implementation of customized probes improves the flexibility in optogenetic experimental design and increases access to wireless probes for in vivo optogenetic research.

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Fig. 1: Design of 3D-POPs.
Fig. 2: Fabrication process for the 3D-POPs.
Fig. 3: Preparation of BLE control module.
Fig. 4: Device integration with a BLE control module.
Fig. 5: Operation of the custom-designed smartphone application for wireless control.
Fig. 6: Surgical procedure for the implantation of a 3D-POP into a mouse brain.
Fig. 7: In vivo behavioral study.

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Data availability

Experiments that support the findings of this protocol are described in our previous paper4. Further data are available from the corresponding authors upon request.

Code availability

The code and STL files used in this study are provided in Supplementary Data 1–3 and 7. Gerber and CAD files for the PCBs are provided in Supplementary Data 4. Code for the BLE SoC and smartphone app are provided in Supplementary Data 5 and 6 and are also available at https://github.com/juulee2011/3DPOPs_control_app_Android.git.

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Acknowledgements

We thank J. Yea, S. Zhang, J. Bilbily and J. Xiao for their help with the initial probe validation. This paper is based on research that has been conducted as part of the KAIST-funded Global Singularity Research Program. This work was also supported by the National Research Foundation of Korea (grant nos. NRF-2021R1A2C4001483 and NRF-2020M3A9G8018572, J.-W.J.), the National Institutes of Health (R01NS117899, R21DA055047, J.G.M.) and the Brain & Behavior Research Foundation (NARSAD YI – 28565, J.G.M.).

Author information

Author notes

  1. These authors contributed equally: Kyle E. Parker, Juhyun Lee, Jenny R. Kim.

Authors and Affiliations

  1. Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO, USA

    Kyle E. Parker, Jenny R. Kim & Jordan G. McCall

  2. Department of Pharmaceutical and Administrative Sciences, University of Health Sciences and Pharmacy in St. Louis, St. Louis, MO, USA

    Kyle E. Parker, Jenny R. Kim & Jordan G. McCall

  3. Center for Clinical Pharmacology, University of Health Sciences and Pharmacy in St. Louis and Washington University School of Medicine, St. Louis, MO, USA

    Kyle E. Parker, Jenny R. Kim & Jordan G. McCall

  4. Washington University Pain Center, Washington University in St. Louis, St. Louis, MO, USA

    Kyle E. Parker, Jenny R. Kim & Jordan G. McCall

  5. School of Electrical Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea

    Juhyun Lee, Choong Yeon Kim, Raza Qazi & Jae-Woong Jeong

  6. Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, Toyohashi, Japan

    Chinatsu Kawakami

  7. Department of Robotics Engineering, Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of Korea

    Kyung-In Jang

  8. KAIST Institute for Health Science and Technology, Daejeon, Republic of Korea

    Jae-Woong Jeong

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Contributions

K.E.P., J.L., J.R.K., J.-W.J. and J.G.M. conceived the project and designed the detailed experimental protocol. K.E.P., J.L., J.R.K., C.K., C.Y.K., R.Q. and K.-I.J. performed the experiments. K.E.P., J.L., J.R.K., J.-W.J. and J.G.M. wrote the paper. J.-W.J. and J.G.M. acquired funding and supervised the project. K.E.P., J.L. and J.R.K. contributed equally to this work. J.-W.J. and J.G.M. are co-senior authors.

Corresponding authors

Correspondence to Jae-Woong Jeong or Jordan G. McCall.

Ethics declarations

Competing interests

J.-W.J. and J.G.M. share two US patents (US 10,617,300 B2 and 11,160,489 B2) for injectable and implantable cellular-scale electronic devices, but do not earn income related to these patents. The other authors have no conflicts of interest.

Peer review

Peer review information

Nature Protocols thanks Ying Fang, Suk-Won Hwang, Chong Xie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Related links

Key reference using this protocol

Lee, J. et al. Adv. Funct. Mater. 30, 2004285 (2020): https://doi.org/10.1002/adfm.202004285

This protocol is an extension to: Nat. Protoc. 12, 219–237 (2017): https://doi.org/10.1038/nprot.2016.155

Extended data

Extended Data Fig. 1 Technical drawing with dimensions for 3D-printed stereotaxic adapter for cannula holder.

a,b, Top (a) and side (b) view of 3D-printed stereotaxic adapter.

Extended Data Fig. 2 Bulk preparation of Metabond quick set cement kit.

a, Selected components of the Metabond kit used to secure the implant in its position. b, Add one scoop of Metabond radiopaque L-powder to the chilled ceramic dish. c, Add four drops of Quick Base liquid. d, Add one drop of Catalyst liquid. e, Mix cement preparation with rounded spatula. f, Use spatula to apply it directly to the skull surface.

Extended Data Fig. 3 Technical drawing with dimensions for a 5-mm-long unilateral 3D-POP and a probe holder.

a, Top view (left) and cross-section view (right) of 3D-POP. b, Top view (left) and cross-section view (right) of probe holder.

Extended Data Fig. 4 Technical drawing with dimensions for a supporter and a stencil mask used for PDMS screen printing.

a, Top view (left) and cross-section view (right) of the supporter. b, Top view of stencil mask.

Extended Data Fig. 5 Circuit diagram for the BLE control circuit.

The circuit includes a BLE SoC, a voltage regulator, indicator LEDs (red, green) and connectors for the integration of a lithium polymer (LiPo) battery in a 3D-POP. The voltage regulator converts fluctuating input voltage from the LiPo battery into constant output voltage (3 V) and supplies it to the BLE SoC. The BLE SoC enables wireless control by smartphone and regulates the photostimulation condition of 3D-POPs. Indicator LEDs help to recognize the communication status of the wireless system.

Extended Data Fig. 6 Preparation of the behavioral assay for optogenetic stimulation.

Preparation for wireless operation. Integrate a 3D-POP and the BLE wireless control module. Check LED operation using the smartphone app. b,c, Preparation for a wired operation: solder wires to a male pin connector (b), and connect a signal generator to a 3D-POP through the wire and apply electrical pulse signals for testing (c).

Supplementary information

Supplementary Data 1

STL file for the stereotaxic adapter

Supplementary Data 2

STL files for the 1D probe

Supplementary Data 3

STL files for PDMS screen printing

Supplementary Data 4

Gerber and CAD files for the wireless control circuit

Supplementary Data 5

Program code for the BLE SoC

Supplementary Data 6

Source codes for the smartphone application

Supplementary Data 7

STL files for the 3 × 3 probe

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Parker, K.E., Lee, J., Kim, J.R. et al. Customizable, wireless and implantable neural probe design and fabrication via 3D printing. Nat Protoc 18, 3–21 (2023). https://doi.org/10.1038/s41596-022-00758-8

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