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Abstract
The MicroLED probe enables optogenetic control of neural activity in spatially separated brain regions. Understanding its heat generation characteristics is important. In this study, we investigated the temperature rise (ΔT) characteristics in the brain tissue using a MicroLED probe. The ΔT strongly depended on the surrounding environment of the probe, including the differences between the air and the brain, and the area touching the brain tissue. Through animal experiments, we suggest an in situ temperature monitoring method using temperature dependence on electrical characteristics of the MicroLED. Finally, optical stimulation by MicroLEDs proved effective in controlling optogenetic neural activity in animal models.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Understanding brain function mechanisms aids in the development of effective medical treatments for neurological and psychiatric disorders, brain machine interface technologies, and novel artificial intelligence technologies based on brain function. As most brain function is not controlled by the neural activity of a single brain territory but is an ensemble of neural activity distributed across multiple territories, measurement techniques spanning multiple brain regions are important. However, since sole utilization of neural activity measurements cannot directly link specific neural activity to behavior, controlling the activity of targeted neural cells with light using genetic modification technologies (optogenetics) is essential for clarifying the role of neural activity in behaviors and associated disorders [1–3]. Due to this, neuroscience tools to control and measure neural activity have been developed for potential worldwide use.
A single optical fiber has been used for optical stimulation, but it is difficult to selectively stimulate different brain regions. It has been reported that a new technology using tapered fibers with optical windows achieved deep multipoint stimulation [4]. However, it still faces challenges in invasiveness, simultaneous multipoint stimulation, and flexibility in multipoint design. Recently, two-photon holographic microscopes for the visualization of hierarchical physiological functions of the cerebral cortex were developed, which enable non-invasive control and measurement of three-dimensional neural activity even in spatially separated regions [5,6]. However, the scattering of light by the cells limits the measurement/control region to an area up to 1 mm from the surface which the laser can reach. Additionally, animal experiments allowing free movement are difficult because two-photon microscope observation require the animals to be immobilized. To provide a solution to this challenge, combination technologies using brain implantation of up-conversion materials and infrared irradiation techniques using a tracking system have been developed, which enables animal experiments with free movement [7,8].
Infrared light cannot easily approach deeper regions, therefore, a method using a scintillator and X-ray irradiation has been reported [9].While such a method using fluorescent materials and excitation light enables optical control of neural activity under free movement, it remains quite difficult to manipulate spatially different neural activities with high temporal resolution to clarify neural networks.
Opto-neural probes using a micro light-emitting diode (MicroLED) are an effective tool to combat this issue [10–17]. The combination of LED devices and wireless communication technologies provides a new method to achieve deep brain stimulation without restricting animal behavior. A needle-shaped Si neural probe integrated with multiple MicroLEDs can facilitate local light stimulation in any region of the hierarchical direction. Notably, a sixteen-MicroLED neural probe could effectively activate local neural activity [17]. By combining the MicroLED neural probes with a neural electrode that can monitor neural activity on multiple channels at the same time, simultaneous observations and control of neural activity in spatially separated brain regions can be conducted, thereby enabling the elucidation of more complex brain neural networks.
Monolithically integrated MicroLED/neural electrode neural probes have been developed [18–20]. Such a probe can simultaneously achieve local optical stimulation and single-unit recording. A major issue in utilizing MicroLED for neuroscience is heat generation in the MicroLED drive. According to the American Association for the Advancement of Medical Devices, an increase in device heat should be minimized to less than 2 °C to avoid brain damage or heat stimulation [21–23]. High light output of the MicroLED is necessary to control the collective neural activity for animal behavior experiments. However, heat generation and light output exhibit a trade-off relationship [24]. Therefore, it is important to understand the relationship between temperature rise and light output in the brain tissue. It has been reported on the LED probe integrated with Pt temperature sensor and the self-sensing method utilizing reverse bias condition of LED [25,26]. Further device development and methodologies are required to accurately monitor the temperature rise during LED drive for in vivo animal experiments.
Therefore, in this study, we investigate the temperature rise (ΔT) characteristics in the brain tissue and have proposed a self-temperature-sensing technique using MicroLED probes. Furthermore, a biological animal experiment, within the allowable temperature rise range, was utilized as a proof of principle to confirm the effectiveness of the MicroLED light stimulation.
2. Experimental methods
2.1 Fabrication of the MicroLED neural probe
The MicroLED neural probe was fabricated using an InGaN-based LED structure with an emission wavelength of 460 nm (ALLOS Semiconductors GmbH, Dresden, Germany) grown on a (111) Si substrate [27]. A mesa structure was formed using inductively coupled plasma-reactive ion etching for n-contact. After forming the photoresist pattern so that the electrode regions were opened by the lift-off process, a SiO2 insulated layer was deposited using the sputtering system. Subsequently, the SiO2 layer on the electrode regions was removed and ITO transparent p-electrode (200 nm) were deposited using an electron beam evaporator and oxygen annealing, followed by the deposition of a Ti/Au n-electrode (50 nm/200 nm) for the n-contact and a metal wiring layer to connect the MicroLEDs. Finally, the needle shaped structure, to smoothly insert the tool into brain tissue, was fabricated by deep-etching the n-GaN layer and Si substrate. The width, length, and thickness of the needle-shaped probe was 3.3 mm, 300 µm, and 300 µm, respectively. Thinner LED probes are required to achieve biological invasiveness for reliable neuroscience research in the future, but this can be achieved by polishing the backside of the substrate. The fabricated probe was mounted on a 15.5 mm × 14.3 mm PCB board. A 1 µm thick Parylene film was deposited as the device encapsulation for bio protection. Figure 1(a) shows photographs of the MicroLED neural probe mounted on a PCB board connected to a compact connector (NSD-36-AA-GS, OMNETICS, Minneapolis, U.S.). The probe consisted of five 50 µm diameter MicroLEDs arranged on a 2 mm region from the tip of the needle. Each MicroLED can be driven independently. For example, bright blue emission from three MicroLEDs was obtained (Fig. 1(a)). Unnecessary parts of the PCB board were removed to avoid interference in animal experiments. The electrical and optical properties of the fabricated MicroLED probes were evaluated using a source meter (6241A, ADCMT, Saitama, Japan) and a power meter (PD300-UV, Ophir Optronics, Jerusalem, Israel). Light output of the MicroLED probe was measured by bringing it as close to the power meter as possible. Figure 1(b) shows current-light output-voltage (I-L-V) characteristics of a representative MicroLED. The clear rectification characteristics were observed at a turn-on voltage of 3.2 V. The light output was approximately 15 mW/mm2 at 1 mA. Considering that a light output of 1 mW/mm2 is required for channelrhodopsin -2 (ChR2) activation [28,29] and a light distribution from the MicroLED [19], the obtained light output was expected to be sufficient for optogenetic stimulation. Also, peak external quantum efficiency (EQE) was approximately 1%, a value similar to that reported in a prior study of an LED integrated on a neural probe [12,17–19]. To confirm the stability of operation in vivo, MicroLED operation was examined in saline. No degradation of light output was observed after one hour of continuous operation at a current of 1 mA in saline. In addition, no degradation of device characteristics was observed after a total of 40 hours of use in animal experiments described in the latter part of this paper. However, long-term chronic experiments would require thicker Parylene coatings to further stabilize LED devices.
Fig. 1. (a) MicroLED neural probe mounted on a PCB board connected to a compact connector and a photograph of the probe tip emitting blue light under a microscope. The probe consists of five MicroLEDs that are 50 µm in diameter with a 2 mm tip. (b) Current-light output-voltage (I-L-V) characteristics of a representative MicroLED. The light output was approximately 15 mW/mm2 at 1 mA corresponding to an external quantum efficiency (EQE) of approximately 1%.
2.2 Animal studies
We confirmed the actual capacity for effective optical stimulation using the MicroLED via animal experiments. All animal procedures were approved by the Animal Care and Use Committee of Dokkyo Medical University and carried out in accordance with the guidelines of the National Institutes of Health. In this experiment, native male mice C57BL6/J (Japan SLC, Inc., Shizuoka, Japan) was used. All mice were maintained on a 12 h light/dark cycle (lights on 7:00 am–7:00 pm) at 24 ± 3 °C and 55 ± 5% humidity, had ad libitum access to food and water, and were housed in a cage with littermates until surgery.
2.2.1 Brain tissue preparation
Mice aged 6-8 months were anesthetized using isoflurane (5%) before decapitation. The brain tissue was extracted from each mouse, the edge was vertically cut by a brain slicer, and was then placed on an aluminum block/temperature control plate to maintain the tissue at 30 °C. The temperature measuring system was then set up around the prepared brain tissue. The prepared tissue was used to record the temperature rise characteristics of the MicroLED.
2.2.2 Optogenetic stimulation of brain CA3 region and recording the local field potential (LFP) from CA1
Surgeries were performed as described previously [30]. The mice aged 6-7 months were anesthetized with isoflurane, administered intraperitoneal injections of medetomidine hydrochloride (0.75 mg/kg of body weight), midazolam (4 mg/kg of body weight), and butorphanol tartrate (5 mg/kg of body weight), and then placed in a stereotactic apparatus (Narishige, Tokyo, Japan). For expressing ChR2(T159C)-EYFP or EYFP in CA3 neurons, AAV9-CaMKII:h:ChR2(T159C)-EYFP or AAV9-CaMKII::EYFP was produced as described previously [31] with pAAV-hCaMKII::ChR2(T159C)-EYFP or pAAV-CaMKII::EYFP plasmid, respectively. The pAAV-CaMKII::hChR2(T159C)-EYFP was constructed by replacing the ChrimsonR-tdT sequence in the pAAV-CaMKII:: ChrimsonR-tdT plasmid (Addgene #99231) with the hChR2(T159C)-EYFP sequence (kindly donated from Prof. K. Deisseroth, Stanford Univ) using the BamHI and EcoRI sites. The pAAV-CaMKII:: EYFP was constructed by replacing the hChR2(T159C)-mCherry sequence in the pAAV-CaMKII::hChR2(T159C)-mCherry plasmid (kindly donated from Prof. K. Deisseroth, Stanford Univ) with the EYFP sequence using the BamHI and BglII sites. For the optical stimulation experiments, 0.3 µL of AAV9-CaMKII::ChR2(T159C)-EYFP (1.79 × 1013 vg/mL) or AAV9-CaMKII::EYFP (2.01 × 1013 vg/mL) was unilaterally injected into the left CA3 region (anterior-posterior [AP], –2.0 mm; medial-lateral [ML], –2.3 mm; dorsal-ventral [DV], –2.0 mm from bregma) using a glass micropipette filled with mineral oil attached to a 10 µL Hamilton microsyringe. A microsyringe pump (Narishige, Tokyo, Japan) and its controller were used to control the speed of the injection (0.1 µL/min). Around 1 month after AAV injection, mice were anesthetized with urethane (1.2 g/kg) and placed in a stereotactic apparatus. The recording tungsten electrode was placed in the right CA1 region (AP, –2.0 mm; ML, + 1.4 mm; DV, –1.3 mm from bregma), and the LED probe was inserted into the left CA3 region (AP, –2.0 mm; ML, –2.3 mm; DV, –2.0 mm from bregma) where ChR2 was expressed at this time. The body temperatures of the mice were maintained by placing them on a heating pad (MK-900; Muromachi Kikai, or ATC-TY; Unique-Medical Inc., Tokyo, Japan) during the recording sessions.
2.2.3 Histochemistry
Mice were deeply anesthetized with 5% isoflurane and an overdose of pentobarbital solution and perfused transcardially with phosphate buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS. The brains were removed and further post-fixed by immersion in 4% paraformaldehyde in PBS for 24 h at 4°C. Each brain was equilibrated in 30% sucrose in PBS and then frozen in dry-ice powder. Coronal sections 30 µm thick were cut on a cryostat and transferred to 12-well cell-culture plates (Corning, Corning, U.S.) containing PBS. After washing with PBS, the floating sections were treated with 4’,6-diamidine-2’-phenylindole (DAPI) (1 µg/ml, Roche Diagnostics, 10236276001) and then washed three times (10 min/wash) with PBS. The sections were mounted on slide glass with ProLong antifade reagents (Invitrogen). Images were acquired using a Zeiss LSM 780 confocal microscope with a Plan-Apochromat 20×, 0.8 numerical aperture, objective lens.
3. Results and discussions
3.1 Temperature rise characteristics of the MicroLED neural probe
Avoiding brain damage and unintended thermal stimulation is important for reliable animal experiments. The ΔT in brain tissue during LED drive can be attributed to the heat generated from the LED device due to emission efficiency and/or heat generation by absorption of the light. There are many reports on ΔT in brain tissue due to optical absorption [32–35]. However, laser power is much larger than the light output generated by MicroLED, which ΔT is 1-2 °C. Therefore, when using MicroLED probe, the ΔT of device determines the ΔT in the brain tissue. In this study, we focused on the ΔT of MicroLED device. The ΔT characteristics in the air and the brain tissue around the MicroLED probe were evaluated using a thermal camera evaluation system (FLIR A325sc, FLIR, Oregon, U.S.) shown in Fig. 2. When measuring the temperature in the brain, the extracted brain was placed on the Al block/temperature control plate. The MicroLED probe was fixed to a manipulator and was smoothly inserted into the brain by adjusting the manipulator. Note that the MicroLED probe surface temperature was observed in the air measurement, however, the brain surface temperature was observed via intracerebral measurement, because the thermal camera measures only surface temperature. To avoid temperature decay in the brain tissue, the MicroLED probe was inserted 50 µm from the edge of the brain tissue. When the LED probe was inserted farther from the edge of the brain, the surface temperature decreased slightly, but no significant change was observed. This indicates that the temperature of the brain tissue near the MicroLED probe could be effectively evaluated. The brain surface temperature did not change before and after inserting the MicroLED probe.
Fig. 2. Configuration of the temperature evaluation system using a thermal camera for the MicroLED probe. Thermal camera was placed on an XYZ precision stage. An extracted brain tissue was placed on an aluminum block/temperature control plate.
Figure 3(a) shows the ΔT in the air and the brain as a function of light output. The MicroLED probe was inserted into the brain tissue up to 1.8 mm depth. The depth was determined assuming optical stimulation of CA3 in the hippocampus in an optogenetic experiment. The MicroLED was driven at 0.2 Hz and a duty ratio of 0.5 from the tip. Light output changed from 8 to 50 mW/mm2, corresponding to the change in current from 0.5 to 4.4 mA. It should be noted that the EQE decreased with the current due to efficiency droop. Both ΔT monotonically increased with increasing light output, however, the ΔT in the brain was approximately one-third that of the air, which may be attributed to the difference in heat dissipation. To confirm that the reason behind the temperature change was heat dissipation, temporal changes in temperature were evaluated, as shown in Fig. 3(b), when the MicroLED was driven for 1 s. The temporal temperature curve in the heat generation process and the cooling process [36] can be expressed as:
where Ts, τth are the saturated temperature, thermal time constant in the heat generation process and cooling process, respectively. Equations (1) and (2) correspond to heat generation and cooling processes, respectively. The temporal temperature curves were well fitted with these equations. The estimated τth showed similar values in both processes, confirming the validity of the fitting method. While the estimated τth in the air was 135-140 ms, the estimated τth in the brain tissue was 500 ms. This suggests that the heat dissipation parameter changed when the probe touched the brain tissue, leading to the ΔT increase. The τth is calculated as the product of heat capacity and thermal resistance. The values of thermal resistance and heat capacity vary greatly between the air and the brain tissue, which explains the change in the thermal time constant [24,37]. However, the heat dissipation parameter depends on not only the material to which heat is transferred but also the area of contact with the brain tissue; that is, the measurement environment.Fig. 3. (a) The ΔT in the air and the brain as a function of light output. (b) Temporal temperature curve in the air and brain tissue when the MicroLEDs were driven for 1 s. The curves were fitted by the equations corresponding to the heat generation process (red line) and cooling process (blue line) to obtain thermal time constants.
To investigate the effect of probe contact area with the brain tissue on the ΔT, the ΔT of brain tissue was evaluated when the penetration depth of the probe was changed from 1.0 to 2.5 mm by altering the manipulator height, as shown in Fig. 2. Figure 4 shows the ΔT and τth as a function of penetration depth. The ΔT decreased from 3.2 to 1.8 °C and τth increased from 280 to 540 ms with increasing penetration depth. However, note that the measurement environment and the position of the heat source (MicroLED) changed at the same time in this experiment because the driving MicroLED was the same. To understand the factor for the determination of thermal time constant, the effect of thermal time constant by driving different MicroLEDs (LED2, LED3, and LED5 shown in Fig. 1(a)) located at the different depth without changing the penetration depth was investigated. As a result, almost no change in thermal time constant was observed. Thus, the change in the thermal time constant is determined not by location of the heat source (MicroLED), but by the measurement environment; that is, the probe contact area with the brain tissue. The probe design and insertion depth required for each targeted neuroscience study varies significantly. Even if a neural probe integrated with a MicroLED with the same characteristics is developed, the ΔT with LED drive would change depending on the brain region. Therefore, in situ monitoring in the brain tissue is important for the effective utilization of the MicroLED probe.
Fig. 4. The temperature rise (ΔT) and the thermal time constant (τth) as a function of penetration depth.
3.2 In situ temperature monitoring using temperature characteristics of the MicroLED
To monitor the ΔT of the MicroLED probe, we focused on the temperature characteristics of the LED. The theoretical temperature dependence of the forward voltage of LEDs [38] can be expressed as:
Fig. 5. (a) Forward voltage as a function of ambient temperature. The temperature coefficient dV/dT was estimated to be –3.4 mV/K. (b) The temporal ΔV and estimated ΔT when the MicroLED was driven at 4.4 mA (50 mW/mm2) for 1 s in the air.
Fig. 6. The ΔT in the brain estimated from current change as a function of light output. The penetration depth of MicroLED probe was 1.8 mm. Inset: Current as a function of ambient temperature. The dI/dT slope changed from 3.8 to 6.4 µA/K with increasing applied voltage. Error bars represent the standard deviation.
3.3 Optogenetic stimulation by MicroLED neural probe
To evaluate the correlation between light output of the MicroLED and optogenetic effects in vivo, optogenetic stimulations in the CA3–CA1 circuit of the mouse hippocampus were performed. In this experiment, two types of mice injected with AAV9-CaMKII::ChR2(T159C)-EYFP or AAV9-CaMKII::EYFP were prepared. As principal neurons of the CA3 region send commissural axonal fibers to contralateral CA1 pyramidal neurons, a MicroLED probe to drive light mediated activation of neurons and tungsten electrode to record LFP were placed at the ChR2-expressing CA3 and contralateral CA1, respectively, as shown in Fig. 7(a) and (b). In this system, it is expected that optical stimulation activates CA3 neurons and then changes the frequency component of LFP of contralateral CA1 in a light pulse dependent manner. The histologic verification of ChR2 expression in CA3 and contralateral CA1 regions in mice used in the experiment was performed (Fig. 7(c)), which indicates that ChR2 was expressed in the CA3 region and axons of CA3 neurons extend to the contralateral CA1 region.
Fig. 7. (a) Photograph of a MicroLED probe being inserted into the brain tissue for optical stimulation. (b) Location of optical stimulation by MicroLED probe and LFP power recording using tungsten electrode. (c) Histologic verification of ChR2 expression in CA3 and contralateral CA1 regions in mouse. Blue: nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI), Green: native fluorescence of green fluorescent protein (GFP). (d) Temporal applied voltage and representative local field potential (LFP) profiles with 20 Hz-optical stimulation in ChR2-EYFP and EYFP mice. (e) Fast Fourier transform analysis of the obtained LFP profiles in ChR2-EYFP and EYFP mice. Power spectral density enhancement (20 Hz) was observed only with optical stimulation of CA3 in mice expressing ChR2-EYFP. (f) Effect of light output on enhancement ratios by optical stimulation in ChR2-EYFP and EYFP mice. Error bars represent the standard deviation of the enhancement ratios obtained from the five optical stimulations.
LED2 placed on the location of CA3 and LED3 placed on the location of outside CA3 were used for optical stimulation. MicroLEDs were driven at frequencies of 4, 8, and 20 Hz and a duty ratio of 0.5 with a light output of 8 mW/mm2 (0.5 mA), corresponding to the ΔT of less than 1 °C. In animal experiments using optical fibers, which are commonly used in optogenetic stimulation, light intensity is performed at several mW /mm2 to several tens of mW/mm2 [41–43]. Therefore, the light output from MicroLEDs applied in animal experiments is a reasonable value. In this study, optical stimulation was performed for 10 s and repeated 5 times with an interval of 1 min, as shown in the top of Fig. 7(d). The representative LFP profiles with 20 Hz-optical stimulation ChR2-EYFP and EYFP mice are shown in Fig. 7(d). Fast Fourier transform analysis of the obtained LFP profiles was performed to confirm the induction of LFP power upon optical stimulation, as shown in Fig. 7(e). Only mice expressing ChR2 stimulated by LED2 showed an increase in power spectral density corresponding to the driving frequency of MicroLEDs at all frequencies. This result indicates that the brain region can be selected for optical stimulation by changing MicroLED.
Finally, the effect of light outputs on LFP power spectra was investigated in ChR2-EYFP and EYFP mice. Optical stimulation was performed at light outputs of 8, 15, 40 mW/mm2 to evaluate the enhancement ratio; which is calculated as the power spectral density during optical stimulation relative to that without optical stimulation. In the case of EYFP mice, the enhancement ratio was almost 1 because no response was obtained even after optical stimulation. In contrast, in ChR2-EYFP mice, optical stimulation resulted in a high enhancement ratio, as shown Fig. 7(f). While a light output of 15 mW/mm2 enhanced the LFP power density compared to the optical stimulation at 8 mW/mm2, the enhancement ratio was unstable for light outputs of 40 mW/mm2, which made plotting difficult in Fig. 7(f). As shown in Fig. 6, a light output of 40 mW/mm2 was expected to increase the ΔT by nearly 3 °C, suggesting that it may have a distinct effect on light-evoked neural activity. Therefore, while this result indicate that the strong light intensity was effective to increase the light-evoked neuronal activation, a higher light intensity can cause thermal problems. We believe that the development of more efficient MicroLEDs and the establishment of methods to utilize MicroLEDs with an understanding of temperature rise will lead to new and improved neuroscience research in the future.
4. Conclusions
In this study, we analyzed heat generation and its impacts in optogenetics applications to effectively utilize MicroLED probes in neuroscience research. The ΔT of the MicroLEDs was determined by changing the heat dissipation parameters corresponding to the surrounding environment. While the ΔT in the brain compared to that in the air decreased, it increased as the probe area touching brain tissue decreased due to changes in penetration depth. This indicated that it is difficult to know in advance the ΔT during animal experiments. Therefore, we investigated the possibility of in situ temperature monitoring using the temperature dependence of electrical characteristics of MicroLEDs. It was confirmed that the forward voltage depends linearly on temperature. The temporal ΔT in the air and the brain were estimated using the obtained value of dV/dT (constant current) or dI/dT (constant voltage), which was in agreement with the results of thermal camera observations, suggesting feasibility of in situ temperature monitoring using the MicroLED. Based on the results of temperature rise obtained from these experiments, the effectiveness of optogenetic stimulation was investigated in a mouse model. The high light output enhanced the light-evoked neuronal activation within the range of driving conditions where ΔT was suppressed. We believe that the establishment of methods for the effective utilization of MicroLEDs will guide the progress of novel neuroscience research.
Funding
Precursory Research for Embryonic Science and Technology (JPMJPR1885); Murata Science Foundation; Takeda Science Foundation; Daiko Foundation; Research Foundation for Opto-Science and Technology; Asahi Glass Foundation; Naito Foundation; Japan Society for the Promotion of Science (21K11556).
Acknowledgements
The fabrication processes were conducted at the facilities of the Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Venture Business Laboratory, and Toyohashi University of Technology.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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