Abstract
This Protocol Extension describes the fabrication and technical procedures for implementing ultrathin, flexible optofluidic neural probe systems that provide targeted, wireless delivery of fluids and light into the brains of awake, freely behaving animals. As a Protocol Extension article, this article describes an adaptation of an existing Protocol that offers additional applications. This protocol serves as an extension of an existing Nature Protocol describing optoelectronic devices for studying intact neural systems. Here, we describe additional features of fabricating self-contained platforms that involve flexible microfluidic probes, pumping systems, microscale inorganic LEDs, wireless-control electronics, and power supplies. These small, flexible probes minimize tissue damage and inflammation, making long-term implantation possible. The capabilities include wireless pharmacological and optical intervention for dissecting neural circuitry during behavior. The fabrication can be completed in 1–2 weeks, and the devices can be used for 1–2 weeks of in vivo rodent experiments. To successfully carry out the protocol, researchers should have basic skill sets in photolithography and soft lithography, as well as experience with stereotaxic surgery and behavioral neuroscience practices. These fabrication processes and implementation protocols will increase access to wireless optofluidic neural probes for advanced in vivo pharmacology and optogenetics in freely moving rodents.
This protocol is an extension to: Nat. Protoc. 8, 2413–2428 (2013); doi:10.1038/nprot.2013.158; published online 07 November 2013
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Acknowledgements
This work is supported by the EUREKA National Institute on Drug Abuse (NIDA) grant R01DA037152 (to M.R.B.), National Institute of Mental Health grant F31 MH101956 (to J.G.M.), and NIDA grant K99DA038725 (to R.A.). We thank the Bruchas laboratory and the laboratory of R.W. Gereau IV for helpful discussions and support. We thank W.Z. Ray for supporting the facilities for the rat surgery. All biomedical aspects of the device work were supported by a National Security Science and Engineering Faculty Fellowship of Energy (to J.A.R.) and startup funding from the University of Colorado Boulder (to J.-W.J.). The LED development was enabled by funding from the US Department of Energy, Division of Materials Sciences, under award no. DE-FG02-07ER46471 (to J.A.R.), the National Institutes of Health Common Fund National Institute of Neurological Disorders and Stroke grant R01NS081707 (to J.A.R. and M.R.B.), and the Materials Research Laboratory and Center for Microanalysis of Materials (grant DE-FG02-07ER46453 to J.A.R.).
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J.G.M., R.Q., G.S., S.L., M.H.I., K.-I.J., Y.L., R.A., and J.-W.J. performed the experiments. J.G.M., M.R.B., J.-W.J., and J.A.R. developed the protocol. J.G.M., M.R.B., J.-W.J., and J.A.R. wrote the manuscript.
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J.A.R. and M.R.B. are co-founders of Neurolux, a company that is making wireless optogenetic probes. The devices described here are not yet part of the company's current product portfolio, but we list this information here as a full disclosure.
Integrated supplementary information
Supplementary Figure 1 Fabrication processes for the ultrathin, flexible microfluidic probe.
(A) Immerse a glass substrate in Pt inhibition solution to activate its surface for easy release of PDMS. (B) Rinse the glass substrate with methanol and bake on a hot plate at 70°C. The glass has amine layer on its surface, which prevents PDMS polymerization. (C) Flip the glass substrate in preparation for PDMS press-casting. (D) Prepare a microfluidic channel mold on a silicon wafer using UV-curable epoxy (SU-8 10). (E) Make anti-stiction surface treatment on the mold wafer by evaporating anti-stiction agent (TMCS) in a completely enclosed wafer box. (F) Cast PDMS on the mold. (G) Press and clip the PDMS-casted mold with the glass substrate and cure it at 70°C for 1 hour. (H) Release PDMS glass substrate from the mold, which has ~20-μm-thick microfluidic channel patterns (channel cross-section = 10 μm ×10 μm). (I–J) Prepare a thin flat PDMS layer on the PC sheet for bonding with the patterned PDMS layer in (H). The thickness of PDMS layer can be controlled by adjusting the spinning speed: 2000 rpm for 60 sec results in 20-μm-thick flat PDMS layer. (M) Treat the surface of both PDMS layer in (H) and (L) with oxygen plasma and bond together. This forms ultrathin PDMS microfluidic channels. (N) Remove PC from the bonded PDMS. (O, P) Release the bonded PDMS layer from the glass substrate. The amine group formed on the glass substrate facilitates delamination of PDMS without damage.
Supplementary Figure 3 Tools used in fabrication of microfluidic neural probes.
(A) Custom-designed blade tool to create a small (500 μm wide) microfluidic probe (Supplementary Data 2). (B) 3D printed alignment tool for punching (Supplementary Data 3). (C) Punch (Harris Uni-Core, 0.50 mm).
Supplementary Figure 4 Schematic diagram showing the operation principle of microfluidic devices for fluid delivery.
By turning on a heater in the device, the thermally expandable layer becomes expanded and pumps out fluid in the reservoir.
Supplementary Figure 7 Circuit diagrams for IR wireless control modules.
(A) Wireless receiver module. (B) Wireless remote controller.
Supplementary information
Combo PDF
Supplementary Figures 1–7 and the Supplementary Note. (PDF 789 kb)
Supplementary Data 1
AutoCAD file for the design of heaters and microfluidic channels. (ZIP 126 kb)
Supplementary Data 2
SolidWorks part document for the custom-designed blade tool to create a 500-μm-wide microfluidic probe. (ZIP 82 kb)
Supplementary Data 3
STL file for the punch alignment tool. (ZIP 8 kb)
Supplementary Data 4
STL file for the device case. (ZIP 47 kb)
Supplementary Data 5
STL file for the case lid. (ZIP 5 kb)
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McCall, J., Qazi, R., Shin, G. et al. Preparation and implementation of optofluidic neural probes for in vivo wireless pharmacology and optogenetics. Nat Protoc 12, 219–237 (2017). https://doi.org/10.1038/nprot.2016.155
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DOI: https://doi.org/10.1038/nprot.2016.155
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