diamond (PCD) substrate for optogenetic stimulation and electrical recording of neural activity. PCD has superior thermal conductivity (up to 1800 Wm-1K-1) [1], ...
A POLYCRYSTALLINE DIAMOND-BASED, HYBRID NEURAL INTERFACING PROBE FOR OPTOGENETICS
Bin Fan1, Ki-Yong Kwon1, Robert Rechenberg2, Anton Khomenko1, Mahmoodul Haq1, Michael F. Becker2, Arthur, J. Weber1 and Wen Li1 1 Michigan State University, MI, USA 2 Fraunhofer USA-CCL, MI, USA shank containing one stimulating site and two recording sites. Finally, the functionality of the PCD probe was demonstrated by recording light-induced action potential from the primary visual cortex (V1) of a channelrhodopsin-2 (ChR2) transfected rat.
ABSTRACT This paper reports a hybrid optoelectronic neural interfacing probe, combining micro-scale light emitting diode (µLED) and microelectrodes on a polycrystalline diamond (PCD) substrate for optogenetic stimulation and electrical recording of neural activity. PCD has superior thermal conductivity (up to 1800 Wm-1K-1) [1], which allows rapid dissipation of localized LED heat to a larger area to improve heat exchange with surrounding perfused tissues, and thus significantly reduce the risk of thermal damage to nerve tissue. During repetitive stimulation with 100ms and 1Hz pulses, the maximum rise in surface temperature of the PCD probe is less than 1 ºC, which is ~90% lower than that of a polymer-based probe. A PCD based probe with two stimulating sites and four recording sites was fabricated. The capacity of the probe for neural stimulation and recording has also been demonstrated in vivo by successfully observing light evoked action potentials.
INTRODUCTION Optogenetics has become a hotspot in the field of neuroscience because it provides the ability to express specific opsins in select neurons, and then use light to modulate their electrophysiological responses [2]. There are several types of light sources for optogenetics, such as incandescent sources [3], laser [4] and micro-LEDs (µLEDs) [5]. Micro-LEDs, in particular, show promise with respect to device miniaturization, simplicity, and low cost of system implementation. Low power µLEDs also have the potential to be integrated with wireless telemetries in order to achieve truly un-tethered systems for studies involving freely behaving animals. However, tissue heating during the operation of µLEDs remains a major challenge. In addition, thermal effects may bias the outcomes of optogenetic experiments, especially when µLEDs are used near tissues receiving continuous light as opsin-negative controls [3]. To address these challenges, we propose a hybrid optoelectronic probe, which utilizes a PCD heat spreader to minimize focal temperature increases during optical stimulation, as shown in Figure 1. Compared to SU-8 that has a thermal conductivity of 0.3 Wm-1K-1 [6], PCD has a thermal conductivity up to 1800 Wm-1K-1. Therefore, the PCD heat spreader of the neural probe can dissipate electrically-induced heat rapidly and uniformly to reduce localized hot spots, thereby minimizing tissue damage during optical stimulation. To validate the efficacy of the PCD heat spreader, the heat distribution and temperature variance of single-shank probes made of SU-8 and PCD were investigated in air using a high-resolution infrared camera. Then, a PCD based probe with two shanks was fabricated, with each
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Figure 1: Concept diagram of the proposed neural probe with a PCD heat spreader.
THERMAL PROPERTY To demonstrate the high thermal conductivity of PCD, a SU-8 probe without a top coating was first fabricated using the method reported in [7], and compared with a PCD based probe with the same dimensions fabricated according to the protocol described later in this paper. A µLED (Samsung, Inc) was mounted onto the probe tip and driven by 1 Hz, 100 ms pulses using different input voltages. Thermal images were taken using a high-resolution infrared camera (Delta Therm HS1570 and DT v2.19 software) and processed using MATLAB® (R2011a, The MathWorks). As shown in Figure 2 (a)-(b), with a 3.4 V input voltage, the SU-8 probe accumulated heat at the tip due to the poor thermal conductivity of SU-8, while the PCD probe dissipated heat throughout the entire shank in less than 0.5 sec without creating a localized hot spot on the probe. Figure 3 (c) shows the cooling curves of the probes after activating the µLED for 60 sec with different input voltages. Figure 3 (d) and (e) show the instantaneous changes in the tip temperatures and steady state temperature variations (after 1 min activation) of the probes for six On-Off cycles, respectively. The maximum temperatures of the SU-8 probe with input voltages of 3.0 V, 3.2 V and 3.4 V increased from 22 ºC to 24.5 ºC, 26.5 ºC and 27 ºC during the first duty cycle (100 mS), continued to increase to 25.5 ºC, 29 ºC and 31 ºC within the first 7 sec, and then stabilized at 26 ºC, 30 ºC and 34 ºC, respectively. On the contrary, the temperature rises of the PCD probe
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with input voltages of 3.0V, 3.2V and 3.4V were within 1 ºC of the baseline temperature. These results demonstrate that application of the PCD probe will not only reduce the risk of thermally-induced tissue damage, but also improve the accuracy of optogenetic experiments by minimizing biological interferences due to thermal effects.
FABRICATION PROCESS The fabrication process is shown in Figure 3. Specifically, (a) PCD was grown on a molybdenum substrate using a 2.45GHz microwave plasma assisted chemical vapor deposition (MW-PACVD) reactor with 2-3kW microwave power in a methane and hydrogen mixture atmosphere (4% CH4, 160-240Torr) and released by thermal stress during cooling from growth temperature to room temperature. (b) The diamond substrate was cleaned by sonication in isopropanol (IPA) and deionized (DI) water for 30 min each and then in nitric acid at 80 ºC for 30 min. (c) A 0.5 µm layer of Cu and 3 nm layer of Ti were deposited using a thermal evaporator (Auto 306, Edward, Inc). Ti was used as an adhesion layer to improve the bonding strength between Cu and PCD. A photoresist mask was patterned using a mask aligner (ABM, Inc) for metal patterning. Then the Cu/Ti layer was wet etched using ferric chloride (to remove Cu) and buffered oxide etchant (to remove Ti) to form microelectrodes, contact pads, and interconnect wires. (d) Photoresist (S1813, Microchem) was spun on and selectively patterned to expose the µLED pad areas. Oxygen plasma (PX-250 plasma system, Nordson March, Inc) at power of 100W and pressure of 0.5Torr was used to remove photoresist residue on the pad areas for 5min. (e) Low melting point (LMP) solder (62 ºC, 144 ALLOY Field’s Metal) was applied in an acid bath [8] and the µLEDs were self-assembled onto the contact pads wetted with LMP solder. (f) Then the substrate was rinsed with acetone, IPA and DI water to remove the photoresist layer. (g) A ~5 µm Parylene C layer was deposited on top of the probe as an encapsulation using a CVD evaporator (PDS 2010, Specialty Coating System, Inc). (h) Parylene C was then patterned using oxygen plasma (RIE-1701, Nordson March, Inc) and a photoresist mask at power of 300W and pressure of 0.25Torr, in order to open the recording sites and contact pads for electrical interconnects. (i) Finally, the probe was shaped using a Nd:YAG laser with power of 8-14W (UltraShape 5xs, Bettonville Inc).
Figure 2: (a)-(b) Heat distribution of the SU-8 and PCD probes. (c) Cooling curves of the probes after activating µLED for 60sec with different inputs. (d) Instantaneous change in the tip temperature. (e) Steady-state temperature variations of the probes.
Figure 3: Fabrication process for making the proposed PCD probe.
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enable the functionality of light sensitivity upon blue light illumination. For viral transduction, the rat was anesthetized with ketamine and xylazine, and then placed in a stereotaxic apparatus. A rostral - caudal incision was made in the skin to expose the skull. Four holes were made medial-laterally with two over each hemisphere of V1. Each site was injected with 1.0 µL (10×1011~10×1012 vector genome (vg/ml) of the virus solution (AAV-hSyn-hCHR2(H134R)-mCherry, UNC Vector Core), with an injection rate of 0.1 µL/min, using a micro syringe (5 µL, Model 75 RN SYR and 100 µL, Neuros Adapter Kit, Hamilton, Inc). After each injection, the syringe was maintained in place for an additional 5 minutes to allow the viral vector to diffuse from around the injection site. Once the injections were completed, the cortical openings were plugged with bonewax, covered with Gelfoam, and the overlying skin was sutured. Then the rat was given 5 µL of sterile saline (0.9% NaCl solution) subcutaneously to prevent dehydration during recovery, as well as an injection of buprenorphine for pain relief. The functionality of simultaneous optical stimulation and recording was tested 3-4 weeks post-surgery to allow for expression of the ChR2 gene in the targeted cortical neurons. Following the same procedure of the viral vector injection, the rat was anesthetized and an opening covering the two injection sites was made for inserting PCD probe. The PCD probe was inserted into V1 of the left hemisphere of the rat with right hemisphere serving as a vector-injected control, as shown in Figure 5 (a). In this case, the µLED on the left side of the probe was driven by repetitive pulses with a frequency of 1 Hz, pulse width of 10 ms, and amplitude varying from 3.2 V to 3.6 V, as shown in Figure 5 (b). Three functioning electrodes were used to record neural activity, through a RHD 2132 32-channels headstage and RHD 2000 USB Evaluation System. The µLED on the right side probe was not tested in this study due to malfunction of the probe before device insertion. Figure 6 (a)-(c) show the neural signals recorded from Channel 2 with different applied voltages. Light-evoked action potentials were observed when the optical stimuli were switched from On to Off with high input voltage of 3.6 V, whereas no action potential was observed with 3.2 V and 3.4 V inputs. The light intensities with 3.2 V, 3.4 V and 3.6 V input voltage were 0.6 mW/mm2, 1 mW/mm2 and 1.5 mW/mm2, respectively. The minimum light intensity to evoke a ChR2 transfected ion channel has been reported to be around 1 mW/mm2 [9]. Considering the coupling efficiency between a µLED and the brain tissue, the input voltage of 3.2 V and 3.4 V of the µLED were not sufficient to evoke any action potential.
Figure 4 (a) shows a micro-fabricated, double-shank probe with one LED and two microelectrodes on each shank. To connect the probe to external powering and recording electronics, the pad areas were covered with the LMP solder, and the thin wires were assembled to the probe, as shown in Figure 4 (b). Epoxy was used to strength the bonding between the pads and wires. The electrochemical impedances of the electrodes at 1KHz (Channels 1-4) were 89.0, 20.6, 38.5, and 54.8 Kohm, respectively. Channel-1 had higher impedance due to incomplete Parylene removal, and therefore was not used in further signal analysis. The dimensions of the PCD probe and µLED are listed in Table 1. The light intensity of the µLED chip was measured using a digital power meter (Model 815 Series, Newport, Inc) and read through the RHA 2000 evaluation board, as shown in Figure 4 (c). 1mm
(a)
40µm
(b)
40µm
1mm
40µm
(c)
Figure 4: (a) A fabricated prototype with close-up views on different segments of the probe. (b) The assembled probe showing that the LEDs were powered up. (c)Light intensity of µLED driven by different input voltages. Table 1 Dimensions of the PCD probe and µLED (L: length, W: width, H: height) µLED (L×W×H)
0.55mm×0.29mm×0.1mm
Shank (L×W)
5mm×1mm
Total dimension (L×W×H)
8mm×6.8mm×0.25mm
Activated µLED PCD probe
IN-VIVO SIGNAL RECORDING In vivo acute experiments were conducted in V1 of a rat to demonstrate the functionality of the as-fabricated PCD probe. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University. Prior to the in vivo testing, the rat was transfected with channelrhodopsin-2 (CHR2) to
Figure 5: (a) In-vivo testing setup. (b) Schematic design of the probe.
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Figure 6: (a)-(c) Neural activity recorded from Channel 2 with µLED input voltage of 3.2 V, 3.4 V and 3.6 V, respectively. (d) A close-up view of signals from Channel 2 with different µLED input voltages. (e) Action potentials recorded from different channels at input voltage of 3.6 V. [3] O. Yizhar, L. E. Fenno, T. J. Davidson, M. Mogri, and K. Deisseroth, “Optogenetics in Neural Systems,” Neuron, vol. 71, no. 1, pp. 9–34, Jul. 2011. [4] A. M. Aravanis, L.-P. Wang, F. Zhang, L. A. Meltzer, M. Z. Mogri, M. B. Schneider, and K. Deisseroth, “An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology,” J. Neural Eng., vol. 4, no. 3, pp. S143–156, Sep. 2007. [5] K. Kwon and W. Li, “Integrated multi-LED array with three-dimensional polymer waveguide for optogenetics,” in 2013 IEEE 26th International Conference on Micro Electro Mechanical Systems (MEMS), 2013, pp. 1017–1020. [6] “SU-8 2000 MSDS.” [Online]. Available: http://www.microchem.com/pdf/SU-82000DataSheet 2025thru2075Ver4.pdf. [7] B. Fan, K. Y. Kwon, A. J. Weber, and W. Li, “An implantable, miniaturized SU-8 optical probe for optogenetics-based deep brain stimulation,” in 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), 2014, pp. 450–453. [8] K. Y. Kwon, B. Sirowatka, W. Li, and A. Weber, “Opto-μ ECoG array: Transparent μECoG electrode array and integrated LEDs for optogenetics,” in 2012 IEEE Biomedical Circuits and Systems Conference (BioCAS), 2012, pp. 164–167. [9] K. Y. Kwon, B. Sirowatka, A. Weber, and W. Li, “Opto- μECoG array: a hybrid neural interface with transparent μECoG electrode array and integrated LEDs for optogenetics,” IEEE Trans. Biomed. Circuits Syst., vol. 7, no. 5, pp. 593–600, Oct. 2013.
CONCLUSION This paper reports the design, fabrication and properties of a PCD-based, hybrid optoelectronic neural interfacing probe, which is capable of optical stimulation and on-chip recording of neural activity. Experimental results show the PCD probes have superior thermal dissipation performance, which not only allows localized LED heat exchange with surrounding tissues, but also minimizing abnormal neural activity due to thermal effects during optogenetic neuromodulation. In vivo testing was performed to demonstrate the functionality of optical stimulating and electrical recording of action potentials of ChR2 transfected neurons. Action potentials were observed with 3.6 V input, 10 ms duration, 1 Hz repetitive pulses. No action potentials were observed with 3.2 V and 3.4 V input. The light intensity testing has proven that only 3.6 V input has exceeded the minimum requirement of opening opsin ion channels, which is 1 mW/mm2. The mechanical properties of the proposed neural probe will be tested in a future study. After the in vivo experiment, the animal was euthanized with an overdose of pentobarbital sodium and the brain tissue perfused and prepared for histological analysis. The gene transfection, actual penetration depth and any tissue injury produced by insertion of the PCD probe will be examined in the future.
ACKNOWLEDGEMENT This work was supported by the National Science Foundation under the Award Numbers CBET-1264772 and ECCS-1407880. The authors would like to thank Dr. Baokang Bi for the help on micro-fabrication and Mr. Yue Guo for the help on drawing the process flow.
REFFERNCES [1] Advanced Diamond Technology, “The CVD diamond booklet.” [Online]. Available: http://www.diamond-materials.com/downloads/cvd_ diamond_booklet.pdf. [2] F. Zhang, V. Gradinaru, A. R. Adamantidis, R. Durand, R. D. Airan, L. de Lecea, and K. Deisseroth, “Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures,” Nat. Protoc., vol. 5, no. 3, pp. 439–456, Mar. 2010.
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