Fabrication and application of flexible, multimodal light-emitting devices for wireless optogenetics

Abstract

The rise of optogenetics provides unique opportunities to advance materials and biomedical engineering, as well as fundamental understanding in neuroscience. This protocol describes the fabrication of optoelectronic devices for studying intact neural systems. Unlike optogenetic approaches that rely on rigid fiber optics tethered to external light sources, these novel devices carry wirelessly powered microscale, inorganic light-emitting diodes (μ-ILEDs) and multimodal sensors inside the brain. We describe the technical procedures for construction of these devices, their corresponding radiofrequency power scavengers and their implementation in vivo for experimental application. In total, the timeline of the procedure, including device fabrication, implantation and preparation to begin in vivo experimentation, can be completed in ~3-8 weeks. Implementation of these devices allows for chronic (tested for up to 6 months) wireless optogenetic manipulation of neural circuitry in animals navigating complex natural or home-cage environments, interacting socially, and experiencing other freely moving behaviors.

Figure 1

Procedure for fabrication of injectable, multifunctional electronics. (a) Thin (~2.5 μm thick) needle-shaped polyethylene terephthalate (PET) is attached on temporary Polydimethylsiloxane (PDMS) coated glass substrate. (b) Schematic and photograph demonstrating the transfer printing of four μ-ILEDs onto the tip of the PET using a PDMS stamp. (c) Passivation approach with photocurable benzocyclobutene (BCB) polymer. The backside of BCB coated substrate is exposed to ultraviolet light. The wave pattern in the upper inset shows ununiformed coating of BCB. The lower inset shows successful uniformed coating. (d) The metal interconnection (Cr/Au) is generated by sputtering, photolithography and metal etching to electrically connect the four μ-ILEDs. (e) The connected device is picked up with water soluble tape. (f) The substrate is separated from the tape after the adhesive is dissolved in the water. The inset shows the μ-ILEDs on freestanding thin, flexible, needle-shaped PET. (g) The device is electrically connected to the ACF cable. The PDMS slabs on top and bottom are compressed using high temperature (~150°C) to bond the ACF cable. (h) The other side of the ACF cable is connected to the PCB with pin connector for wireless or wired powering schemes. (i) The ACF cable and PCB is coated with PDMS for waterproofing. (j) Blue (450 nm) μ-ILEDs are powered. The μ-ILEDs and electrical connection should be checked prior to injection (k) The device is assembled with injection μ-needle using biodissolvable silk adhesive. (l) Image of a completed device ready for injection into brain tissue.

Figure 2

Multifunctional sensors and optoelectronics. (a) Representative scheme for multifunctional, injectable electronics formed on injectable needle. The devices include electrophysiological sensor (μ-electrode); 1^st layer), silicon photodiode (μ -IPD; 2^nd layer), four microscale inorganic light-emitting diodes (μ-ILEDs; 3^rd layer), and temperature sensor (μ-temp. sensor; 4^th layer) based on platinum resistor are formed on injectable μ-needle fabricated from epoxy polymer. (b) Side view of such a device reveals the ultrathin nature of the active components of the device. Scale bar applies to both panels and = 200 μm.

Figure 3

Wireless operation and equipment. (a) An experimental setup for wireless power transmission. The setup contains a RF signal generator (1), a RF power amplifier (2), a DC power supply (3), and a panel antenna (4). Components for the wireless power harvester for μ-ILED powering with stacked PCB circuits (5: circuit contains a ceramic antenna and a capacitor is connected between the feed line of the antenna and the ground plane to match the impedance of the antenna with the next circuit. 6: a second circuit contains a voltage multiplier constructed with 6 pairs of capacitors and Schottky diodes in a cascaded connection).before (b) and after (c) connecting with copper wire. A completed wireless power harvester alone (d) and with (e) connection to μ-ILED device (7) for wireless operation. Top (f) and side views (g) of a flexible wireless power harvester on Kapton film with similar components as the wireless harvester on PCB circuits. (h) A completed flexible wireless power harvester with connection to μ-ILED device for wireless operation. All scale bars are 5 mm. (i) A schematic of the PCB-based power harvester. The numbered circuit components correspond to the same number shown in Fig. 3b and 3e. (j) A schematic of the wireless power harvester.

Figure 4

Surgical procedure for injection of virus and μ-ILED devices into mouse brain. (a) Custom-built adapter for accurate stereotaxic placement of device (see EQUIPMENT SETUP). (b) Mounted μ-ILED device, ready for injection into the animal. The exposed μ-needle is grasped with the adapter and a small piece of tape is used to secure the PCB during surgery. (c) A properly mounted mouse with head shaved and eyes-lubricated is ready for surgery. (d) Betadine and ethanol is used to prevent infection and the scalp is open to expose the skull. (e) After leveling the skull, the drill is used to create pilot holes for the bone screws. (f) Forceps and a spatula or jewelry screwdriver is used to drive the screws into the skull. (g) The syringe needle is lowered to the desired coordinates to deliver the virus containing the optogenetic construct. (h) A μ-ILED device prepared to be driven into the brain using the same craniotomy as the viral injection. Dashed lines outline the shape of the device for clarity. (i) The μ-ILED device is lowered into the tissue and ACSF is applied to the skull surface to dissolve any external silk adhesive. (j) After a 15 minute waiting period, the μ-needle is carefully retracted from the skull. (k) Dental cement is applied directly to the craniotomy site to secure the μ-ILED device in its targeted position. (l) The PCB connector is secured above the bone screws using a second layer of dental cement. (m,n) The PCB connector is completely encapsulated in dental cement, taking care to ensure that no bonds are made directly to the soft tissue. (o) The adapter is shown following surgery, containing only the μ-needle.

Figure 5

Expected results following viral and device injection. Once a device is injected, the standard connection allows for temporary coupling multiple means of powering in a variety of behavioral assays: (a) A mouse connected for wired powering in a standard operant behavioral chamber, (b) the same mouse prepared for wireless powering using the lightweight, flexible power scavenger in a conditioned place preference environment, (c) Two mice with implanted devices amongst cage mates. The mouse in the foreground has a PCB-style RF scavenger for powering in a homecage environment.

Figure 6

μ-ILED device recycling and refabrication for subsequent use. (a) The same headcap from Fig. 4, removed from the animal post-mortem and cleaned of biological material. (b) The headcap should then be fully submerged in methyl methacrylate. (c) Following overnight incubation in the stabilized methyl methacrylate monomer, the PCB, connector, μ-ILED device, and bone screws will be freely available in the solution. (d,e) Both connections with the ACF cable will also dissolve, rendering the device non-functional. (f) The device should be checked for reusing. If non-functional, the device should be discarded and a new device should be fabricated. (g) The working device is reassembled with new ACF cable and PCB. (h) The electrical connection through the new ACF cable should be checked after the device is attached with injection μ-needle again.