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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Prog Biophys Mol Biol. 2020 Dec 24;162:89–100. doi: 10.1016/j.pbiomolbio.2020.12.004

Infrared neural stimulation at different wavelengths and pulse shapes

Yingyue Xu 1,2, Mario Magnuson 1, Aditi Agarwal 1, Xiaodong Tan 1, Claus-Peter Richter 1,2,3,4
PMCID: PMC8905667  NIHMSID: NIHMS1662251  PMID: 33359901

Abstract

Neural stimulation with infrared radiation has been explored for brain tissue, peripheral nerves, and cranial nerves including the auditory nerve. Initial experiments were conducted at wavelengths between λ=1,850 and λ=2,140 nm and the radiant energy was delivered with square pulses. Water absorption of the infrared radiation at λ=1,860 nm is similar to absorption at wavelengths between λ=1,310 and λ=1,600 nm, which are in the radiation wavelength range used for the communication industry. Technology for those wavelengths has already been developed and miniaturized and is readily available. The possibility of the infrared light to evoke compound action potentials (CAP) in the cochlea at λ=1,375, λ=1,460, and λ=1,550 nm was explored and compared to that of λ=1,860 nm in guinea pigs. Furthermore, rise and fall times of the 100 μs long pulses were changed and four basic pulse shapes (square, triangular, ramp-up, and ramp-down) were explored in their ability to evoke a CAP. In animals with pure tone threshold averages (PTAs) above 70 dB SPL, the results show that the favorable wavelength is λ=1,460 nm to reach threshold for stimulation and λ=1,375 nm or λ=1,460 nm for obtaining maximum amplitude. The most favorable pulse shape is either ramp-up or triangular.

Introduction

Neural prostheses deliver electrical current to nerves or neural tissue to restore lost function or to modulate neural activity for treatment. The quality of the treatment depends on the precision by which the stimulation can be achieved. Determined by the physical properties of the tissue, the electrical current spreads widely and precise neural stimulation has not been possible, which limits the success of the treatment. The current spread is a challenge for all prosthetic and neuromodulation devices. In contrast to electrical current, a pulsed laser was used to selectively activate small segments of the rat sciatic nerve (Wells et al., 2005a; Wells et al., 2005b) or individual branches of the facial nerve (Teudt et al., 2007). Selective stimulation with infrared radiation has also been shown for the auditory system (Moreno et al., 2011; Rajguru et al., 2011; Richter et al., 2011a) and the vestibular system (Rajguru, 2018). With optical stimulation, it is anticipated that neural prostheses with enhanced neural fidelity can be developed.

Neural stimulation with infrared radiation works through spatially and temporally confined heating of the tissue. Upon the absorption of the photon by the water, its energy is converted into a temperature change, which then evokes an action potential (Albert et al., 2012; Okunade et al., 2013; Plaksin et al., 2017; Shapiro et al., 2012; Wells et al., 2007a; Yao et al., 2009). While a single mechanism for INS has not been identified, a series of events that occur at the same time lead to an action potential of the irradiated nerve. In the following, some of the identified effects of infrared radiation on the nerve are summarized. Spatially and temporally confined heating depolarizes the cell by changing the membrane capacitance (Liu et al., 2013; Okunade et al., 2013; Plaksin et al., 2017; Rabbitt et al., 2016; Shapiro et al., 2012) resulting in a depolarizing inward current. The change in capacitance might result from changes in membrane thickness (Plaksin et al., 2017) or from small-diameter nanopores in the membrane (Beier et al., 2014). Furthermore, it has been shown that transient receptor potential cation channels of the vanilloid group (TRPV) are activated (Albert et al., 2012; Barrett et al., 2018; Rhee et al., 2008; Suh et al., 2009; Yao et al., 2009). They are temperature sensitive and are highly calcium selective (Baylie et al., 2011; Güler et al., 2002; Gunthorpe et al., 2002; Jia et al., 2007; Kauer et al., 2009; Lee et al., 2005; O’Neil et al., 2005; Santoni et al., 2011; Sharif-Naeini et al., 2008; Sladek et al., 2013). Studies on isolated neurons demonstrated that intracellular calcium concentration increases during INS resulting in a depolarization of the neuron (Dittami et al., 2011; Lumbreras et al., 2014; Lumbreras et al., 2013; Rajguru et al., 2010; Rajguru et al., 2011). Spatially and temporally confined heating, which occurs during INS, also results in stress relaxation waves (Baumhoff et al., 2018; Kallweit et al., 2016; Schultz et al., 2012a; Schultz et al., 2012b; Teudt et al., 2011; Thompson et al., 2015), which are able to mechanically stimulate remaining hair cells in the cochlea.

A spatially confined temperature change is the first step in the sequence of evoking an action potential with infrared radiation (Liu et al., 2014; Okunade et al., 2013; Plaksin et al., 2017; Rabbitt et al., 2016; Shapiro et al., 2012; Wells et al., 2007a). This heating, however, also bares the risk for thermal damage to the tissue (Goyal et al., 2012; Wells et al., 2007b). While previous experiments have used square pulses and a wide range of wavelengths in the infrared to stimulate the auditory systems, it remains of interest, which selected wavelengths are optimal and whether the time over which the heating occurs is critical. With this paper we will address the questions if stimulation of neurons with infrared radiation can be optimized by through selecting a specific radiation wavelength and by using different pulse shapes.

Methods

All procedures followed the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Northwestern University.

Animals

Eleven Hartley guinea pigs were purchased from a commercial breeder, Kuiper Rabbit Ranch (Gary, IN), including seven males and four females. The age of the animals was between 4 weeks and 6 months, their weight between 597 and 1290 gram (g).

Animal anesthesia, surgery and electrode placement

After the guinea pigs arrived at Northwestern University, the animals were allowed to adjust to their new environment for at least 2 weeks. Anesthesia and surgical methods followed a previously published approach (Richter et al., 2011b; Tan et al., 2015; Young et al., 2015). During the terminal experiment, they were anesthetized with 0.9 mg/kg urethane dissolved in a 0.1 molar saline solution. Following the urethane injection, the level of anesthesia was then adjusted with isoflurane (0–1%) and nitrous oxide (49%) in oxygen. After the animals were anesthetized, a tracheotomy was made and a plastic tube was secured into the trachea to facilitate breathing. Throughout the experiment, the animals were ventilated with oxygen using an anesthesia workstation (Hallowell EMC, Pittsfield, MA). Depth of anesthesia was assessed every 15 minutes by a paw withdrawal reflex. Core body temperature was maintained at 38°C with a thermostatically controlled heating pad. Heart rate, respiration rate, body temperature and O2-saturation were monitored continuously using a Bionet BM3 vet (Bionet Co. Ltd, Seoul, Korea) monitoring system and were logged in 15-minute intervals.

After each guinea pigs was anesthetized, its head was fixed with dental acrylic (Methyl Methacrylate, CO-ORAL-ITE DENTAL MFG CO, Diamond Springs, CA) to a custom-made head holder, using three 1.5 mm stainless steel self-tapping cortex screws (Veterinary Orthopedic Implants, St. Augustine, FL) as anchors. The left cochlea was surgically accessed through a post auricular “C”-shaped skin incision. Cervicoauricular muscles were removed and the outer ear canal was exposed. For easier placement of the speaker with its speculum, the cartilaginous outer ear canal was sectioned. The bulla was exposed and opened about 2 × 2 mm with a motorized drill (World Precision Instruments, Sarasota, FL). A cochleostomy was made approximately 0.5 mm from the round window with the same drill (Figure 1). A silver electrode was made from a 125 μm Teflon insulated silver wire (A-M systems, 131 Business Park Loop Sequim, WA) and was placed on the RW membrane to record compound action potentials (CAPs) during acoustic and optical stimulation.

Figure 1.

Figure 1.

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The x-ray image shows a 200 μm optical fiber inserted through a cochleostomy into the basal turn of the guinea pig cochlea. Scale bar equals 2 mm.

Acoustic stimuli

Acoustical stimuli were pure tone bursts (12 ms in duration, including rise and fall times of 1 ms). The first frequency for the pure tone stimuli was 32 kHz. It was decreased in 2 steps per octave over 6 octaves. Sound levels at each frequency began at the loudest speaker output and were attenuated in steps of 5 dB. The voltage commands for the acoustic stimuli were generated using custom software written in TestPoint® and were delivered at a rate of 5 Hz to a Beyer DT770-Pro headphone. The sound level was calibrated with a Brüel and Kjær 1/8-inch microphone as described before (Cheatham et al., 2001; Pearce et al., 2001).

Optical Stimulation

Optical stimulation was achieved with four different diode lasers. The diode laser emitting at λ=1,860 nm was from Lockheed Martin Aculight Corp. (Bothell, WA) and was the same laser used in our previous experiments. Its pulse shape was limited to a square pulse. The pulse duration was 100 μs and pulses were delivered at a rate of 5 pulses per second (pps). The rise and fall time of the laser pulse was 3 μs. Three diode laser modules from SemiNex (Peabody, MA) were used. They emitted radiation at different wavelengths, λ=1,375 (4PN-123), λ=1,460 (4PN-101), and λ=1,550 nm (4PN-108). The pulse duration was 100 μs. Pulses were delivered at about 5 pps. While faster pulse repetition rates are possible, the slow pulse rate was selected to avoid interactions between subsequent pulses when measuring the CAPs. The radiant energy per pulse was controlled through a High-Power Precision Laser Diode Driver (LDX32420, ILX Lightwave, Bozeman, MT). The energy was measured in air at the tip of the optical fiber using the J50LP-1A energy sensor (Coherent, Santa Clara, CA). The results from the laser calibration are shown in Figure 2. The output of the lasers was coupled to a 200-μm flatpolished optical fiber (P200–5-VIS-NIR, Ocean Optics, Dunedin, FL, NA=0.22), which was inserted through the cochleostomy into scala tympani of the basal turn of a guinea pig cochlea to deliver the infrared radiation to the target tissue (Figure 1). Note that polishing the optical fiber is crucial for a homogenous irradiation area, after the fiber has been immersed into fluids. The spot size at the target was estimated previously from the neural responses in the inferior colliculus to 360 μm (Richter et al., 2011a).

Figure 2.

Figure 2.

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Lasers were calibrated prior to the experiments by measuring their radiant energy at different current levels. Four pulse shapes were measured: square pulse (black circles), triangle pulse (green squares), ramp up (blue triangles) and ramp down (red diamonds). The energy was plotted along the current levels. A linear fitting was performed for current levels and energy for each pulse shape. The energy at low current levels was predicted with the linear fitting. The laser emitting at λ=1,860 nm could only deliver square pulses.

One of the independent variables was the laser rise and fall time. For square pulses, the time was 10 μs, which was determined by the laser diode driver. For the ramp-up the rise time and for the ramp-down waveforms the fall time was 90μs. Likewise, for the ramp-up pulse shape the fall time and for the ramp-down pulse shape the rise time was 10 μs. For the triangular wave, the rise and fall time was 50 μs. One outcome measure was the compound action potential amplitude, which was measured during stimulation with different pulse shapes.

Pressure in the outer ear canal (oEC)

Pulses of infrared radiation, which are delivered with an optical fiber into the fluid filled scala tympani of the guinea pig cochlea result in a transient heating of the target volume and the generation of a pressure wave. The laser-induced pressure is propagated back through the middle ear into the outer ear canal where it was measured with a calibrated sensitive microphone. The microphone is part of the distortion product otoacoustic emission system ER10C (Etymotic Research, Elk Grove Village, IL). For the pressure measurements the tip of the ER-10C was inserted into the cartilaginous outer ear canal of the guinea pig and sealed with a self-expanding foam tip. The ER-10C was set to 40 dB amplification and microphone responses to 100 stimuli were captured and averaged with a personal computer equipped with a KPCI-3110 (Keithley, Cleveland, OH) analog-to-digital computer board at a rate of 250 kHz while laser pulses were delivered to the cochlea. The maximum amplitude determined from the recorded traces served to calculate the peak pressure in the outer ear canal (Figure 3). Note that the frequency spectrum of the ER-10C is limited to frequencies below 20 kHz.

Figure 3.

Figure 3.

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The image shows a trace of the microphone response during laser stimulation in the cochlea. The laser pulse was a square pulse delivered at 4 ms. The radiant energy was 110 μJ/pulse.

From the measured sound level in the ear canal one can make several predictions by using the forward and reverse middle ear transfer function. The gain for the forward transfer function in the guinea pig has been measured by others and is about 25 dB. The loss of the reverse transfer function is about 35 dB (Magnan et al., 1997; Magnan et al., 1999). Hence, the pressure during laser stimulation is 35 dB lower in the outer ear canal than in the cochlea (Xia et al., 2018). Consequently, the sound level from a speaker must be 10 dB higher than the level measured during cochlear laser stimulation to generate a similar pressure in the cochlea with an acoustic stimulus delivered to the outer ear canal (Xia et al., 2018).

Damaging of the cochlea

The cochleae were damaged by a single transtympanic injection of about 250 μL of Neomycin (25 mM) in Ringer’s Lactated Solution (RLS). For the injection, the animals were sedated with isoflurane 3% in oxygen, given over a facemask. After the procedure, the animals were recovered from anesthesia and allowed to survive for at least 4 weeks for the damage and neural degeneration to occur. Cochlear function was assessed prior to each of the experiments with acoustic stimulation and optical radiation.

Outcome measures

Compound action potentials (CAPs)

CAPs were measured at the round window during cochlear stimulation. The CAP recording electrodes were connected to a differential amplifier (ISO-80, WPI, Sarasota, FL) with a high-input impedance (> 1012 Ω) set at a gain of 60 dB. The signal was filtered using a band pass filter, 0.3 to 3 kHz (filter slope −12dB/octave). Responses to 50 stimulus presentations were averaged and saved. The sampling rate was 250 kHz. For acoustically evoked CAPs, recordings were taken before and after creating the cochleostomy and at several times between the sets of stimulation with the laser to monitor cochlear function. The pure tone average (PTA) threshold was calculated as the average threshold at 2, 4, 8, 16, and 32 kHz.

Data Analysis and Statistics

Determine CAP amplitude and latency

Analysis was done using custom written software in MATLAB and through visual inspection of each of the traces. The CAP amplitude was the difference between the first minimum (N1) and the first maximum (P1) following the stimulus. CAP amplitudes were plotted versus different sound levels and radiant energy levels. The latency of the N1 was also determined and plotted as a function of the radiant energy per pulse, at 30 μJ/pulse, and for an N1-amplitude of 50μV.

CAP amplitudes vary among the animals. To better demonstrate response patterns, we normalized the CAP amplitudes. To compare the efficacy of infrared stimulation at λ=1,860 nm and square shaped pulses, we normalized the data by dividing each value obtained in an animal by the corresponding value at λ=1,860 nm. The data were then pooled into data obtained in guinea pigs with PTA thresholds above 70 dB SPL and guinea pigs with PTA thresholds below or equal to 70 dB SPL (Figure 4). The 70 dB SPL criterion was based on the pressure measured in the ear canal during INS at 100μJ/pulse and on the slope of the N1-latencies, the time between the presentation of the optical stimulus and the peak of N1. Data were averaged before and after normalization and the corresponding standard error was calculated.

Figure 4.

Figure 4.

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Shown are the sound levels required for visible CAP amplitude (CAP threshold) evoked by pure tone stimuli at 32, 16, 8, 4, and 2 kHz. Guinea pig 1 and 2 (green traces) have a pure tone average threshold (PTA) below 50 dB SPL (re 20 μPa). Guinea pig 3 through 6 (blue traces) have PTAs between 50 and 70 dB SPL. The threshold elevation is in particular above 8 kHz. Three animals have no response to acoustic stimuli at 32 kHz. Guinea pigs 7 through 11 (black traces/markers) have PTAs above 70 dB SPL. The red horizontal line provides the sound level recorded in the outer ear canal while laser pulses at 100 μJ/pulse was delivered to the cochlea. Note, the radiant energy to reach stimulation threshold is about 10 times less.

Statistical analysis

Descriptive statistics were applied. Examples of a typical experiment as well as population data are shown. The recorded values are plotted versus the radiation energy delivered to the cochlea. Statistical analysis on data sets with multiple variables is challenging because of the possible inherent correlations: across ears, time, and frequency. We executed an analysis of variance (ANOVA). In case the ANOVA was significant, we used the Tukey honestly significant difference test to determine whether the differences between the groups are significant.

Results

Hearing assessment

Cochlear function was assessed by CAP recordings. The sound level required for a visible CAP response in the recorded traces was determined at 32, 16, 8, 4, and 2 kHz and is shown in Figure 4. Missing markers indicate that no response could be evoked with acoustic stimuli up to the highest sound level produced by the speaker.

Two groups of animals were formed based on their PTA thresholds. Group one included normal hearing animals (green traces; PTAs ≤ 50 dB SPL; N=2) and animals with moderately elevated thresholds (blue traces; PTAs > 50 dB SPL and ≤70 dB SPL; N=4). Group two had animals with severely elevated thresholds (black traces: PTAs >70 dB SPL; N=5). In case that no response to acoustic stimuli was recorded at a given stimulus frequency, the maximum sound level plus 10 dB was used at this frequency to calculate the PTA threshold.

N1-latencies

Figure 5 shows the increase in CAP amplitude with increasing radiant energy per pulse. The example in Figure 5A is for a hearing animal and in Figure 5D for an animal with elevated thresholds. The trace and marker colors are for different wavelengths, λ=1,375 (black circles) λ=1,460 (blue diamonds) λ=1,550 (green triangles), and λ=1,860 nm (red circles). For the hearing animals (Figure 5A–C) the N1-latency decreases with increasing radiant energy (Figure 5B) and N1-amplitude (Figure 5C). This is different in the animals with elevated CAP thresholds (Figure 5D–F). The N1-latency appears constant for increasing radiant energies (Figure 5E) and N1-amplitudes (Figure 5F). The slope for the N1-latency change was calculated and plotted in Figure 5G. It is smaller in animals with elevated threshold than in hearing animals. The vertical line in the plots identifies a PTA of 70 dB SPL. The N1-latencies at 30 μJ/pulse (Figure 5H) and at an N1-amplitude of 50 μV (Figure 5I) change systematically with the hearing status. Normal hearing animals have latencies of about 1.8 ms, while animals with severely elevated thresholds have latencies of about 2.8 ms (Figure 5I).

Figure 5.

Figure 5

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(A) shows the CAP amplitude increase with increasing radiant energy per pulse in a hearing animal with a PTA of 31.4 dB SPL and (C) shows the same plot for a deaf animal with a PTA of 61.6 dB SPL. (B) and (E) show the latency for the N1 from the stimulus. The latency decreases with increasing radiant energy from about 2 ms to about 1.6 ms in hearing animals and is constant in hearing impaired animals. Similar behavior can be seen if the latencies are plotted versus the N1-amplitude as shown in (C) and (F). From the plots shown in (C) and (F), the slope for the latency was calculated and is shown in (G). Panels (H) and (I) show the latencies for the guinea pigs with different hearing status as expressed by the PTA. Latencies are shown for a radiant energies of 30μJ/pulse (H) and for an N1-amplitude of 50 μV. Latencies are typically larger for deaf animals. The trace and marker colors are for the different wavelengths, λ=1,375 (black circles) λ=1,460 (blue diamonds) λ=1,550 (green triangles), and λ=1,860 nm (red circles).

Wavelength differences

Irradiation with infrared light leads to heating of the target volume and subsequent generation of stress relaxation waves or thermal induced pressure waves. To determine whether irradiation at different wavelengths in the cochlea results in acoustic events in the outer ear canal, pressure levels were measured for the laser parameters used in the experiment. Figure 6 shows that no differences in the pressure exist for different wavelengths or pulse shapes. The pressure was similar in the outer ear canal of all animals, including those with elevated thresholds and animals with normal cochlear function.

Figure 6.

Figure 6.

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(A) and (B) show examples for the pressure measured with ER10C in the ear canal, (A) for different wavelengths and (B) for different pulse shapes (the radiation wavelength in (B) is 1,375 nm, the pulse length 100 μs, the optical fiber core diameter 200 μm. (C) shows the cumulative data for the differences in the pressure in the ear canal at different wavelengths obtained for a 100 μs-square pulse, at 30 μJ/pulse). Differences are small and statistically not significant. (D, E, and F) show the cumulative data for the differences in the pressure in the ear canal for different pulse shapes, square (sq), triangle (tr), ramp-up (ru), and ramp-down (rd). pulses were 100 μs in duration and the radiant energy was 30 μJ/pulse. Pressure was similar for the different pulse shapes. On each box, the central mark indicates the median, the bottom and top edges of the box indicate the 25th and 75th percentiles, and the whiskers extend to the most extreme data points not considered outliers.

CAPs were evoked with radiation at wavelengths of λ=1,375 (Figure 7, red trace), λ=1,460 (Figure 7, blue trace), λ=1,550 (Figure 7, green trace), and λ=1860 nm (Figure 7, black trace) delivered with square shaped pulses using an optical fiber placed through the cochleostomy into scala tympani. The light beam was directed towards the spiral ganglion. Connecting the input of the optical fiber to the output of the different lasers was used to change the wavelength. The tip of the optical fiber in the cochlea was not changed in position between measurements. At each wavelength the CAP responses at increasing radiant energies were recorded. The CAP amplitude first rapidly increases, and then saturates at higher radiant energies.

Figure 7.

Figure 7.

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The figure shows the responses obtained from a guinea pig with a PTA of 80.6 dB SPL. The traces show the CAP peak-to-peak amplitude at different radiant energies for four wavelengths, λ=1,375 (red), λ=1,460 (blue), λ=1,550 (green), and λ=1,860 nm (black). The pulse shape was a 100 μs-square pulse.

The population data are shown in Figure 8. Green markers show results from the animals in group 1 and red markers the results from the animals in group 2. Figure 8A shows the radiant energy to reach stimulation threshold and Figure 8B the CAP amplitude during stimulation at 40 μJ/pulse. The energy was delivered with square shaped pulses. The data variation is large and it is difficult to determine true wavelength dependencies. To determine changes, the data was normalized by dividing the values for threshold radiant energy and CAP amplitude at each radiation wavelength by the corresponding value obtained for λ=1860 nm. The results are shown in Figures 9A and 9B. Animals from group 2 had the largest CAP response amplitude when stimulated with triangular shaped pulses at λ=1,375 nm (Figure 9).

Figure 8.

Figure 8.

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(A) shows the thresholds for stimulation obtained for all animals, (B) shows the CAP amplitudes for stimulation obtained during stimulation with square pulses at 40μJ/pulse measured for all animals. Green markers are for animals with pure tone averages (PTAs) <70 dB SPL and red markers for animals with PTAs >70 dB SPL. Diamond markers with the error bars are the averages ± standard errors.

Figure 9.

Figure 9.

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Averaged responses (mean ± standard error) obtained from the animals in group 2 obtained at different wavelengths and with different pulse shapes were compared to the responses obtained for λ=1,860 nm using square shaped pulses. (A) shows the ratios for the radiant energy required for stimulation, (B) shows the CAP amplitudes obtained during irradiation with pulses at 40μJ/pulse. Thresholds were lowest at λ=1,460, λ=1,550, for triangular and ramp-up shaped pulses and at λ=1,860 nm for square shaped pulses. CAP response amplitudes at 40μJ/pulse were in general larger for λ=1,375.

The average CAP response amplitude at 40 μJ/pulse for animals in group 1 was 106.9±24.4, 95.2±16.5, 61.6±9.7 and 106.6±36.1 μV at radiation wavelengths of λ=1,375, λ=1,460, λ=1,550, and λ=1,860 nm, respectively. The ANOVA was not significant (DF=23, F=0.81, Fc=3.71, P=0.51). The average CAP response amplitude for the animals in group 2 was 22±11, 21±6.1, 22±7.3 and 30±8.4 μV at radiation wavelengths of λ=1,375, λ=1,460, λ=1,550, and λ=1,860 nm, respectively. The ANOVA was not significant (DF=18, F=0.23, Fc=3.29, P=0.88). The average thresholds for stimulation for animals in group 1 were 10.5±2.1, 11.2±2.8, 12.4±4.8, and 9.5±2.7 μJ/pulse at λ=1,375, λ=1,460, λ=1,550, and λ=1,860 nm, respectively. The ANOVA was not significant (DF=23, F=0.14 Fc=3.1, P=0.94). For animals of group 2 the average threshold was 32.3±9.9, 30.2±8.5, 37.4±12.7, and 41.3±22.2 μJ/pulse. The ANOVA was not significant (DF=18, F=0.14, Fc=3.3, P=0.94).

Pulse shape differences

The radiant energy required for a CAP response evoked by square, triangular, ramp-up, and ramp-down shaped pulses were tested for each of the wavelengths. An example for CAPs evoked with optical pulses of different rise and fall time at λ=1,375 nm is shown in Figures 10. It appears that ramp-up pulses are more efficient to evoke a CAP.

Figure 10.

Figure 10.

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CAPs evoked by four different pulse shapes at the wavelength of λ=1,375 nm. (A) shows the CAPs generated by square shaped pulses. (B) shows CAPs generated by triangle shaped pulses. (C) shows CAPs generated by ramp up shaped pulses. (D) shows CAPs generated by ramp-down shaped pulses.

Figure 11 shows the results obtained in a different animal to this shown in Figure 10. Shown are CAP response amplitudes to pulses with different shapes (square, triangle, ramp-down, and ramp-up) at increasing radiant energy per pulse with radiation wavelengths of λ=1,375 nm (Figure 11A), λ=1,460 nm (Figure 11B), and λ=1,550 nm (Figure 11C). In each example the response amplitude grows rapidly after reaching threshold for stimulation and saturates at high radiant energy levels. Note, the waveform of the laser emitting at λ=1,860 nm could not be modified and therefore, data were only acquired for square shaped pulses.

Figure 11.

Figure 11.

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The response amplitude to optical stimulation in the cochlea depended on the pulse shape of the optical radiation. Data from different pulse shapes are coded by the following marker shapes: square pulse = squares, triangular pulses = triangles, ramp-down pulses = circles, and ramp-up pulse = asterisks. (A) shows an example for the CAP peak-to-peak amplitude at λ=1,375 nm, (B) at λ=1,460 nm, and (C) at λ=1,550 nm. For the given example, ramp-up shaped evoked the largest responses, and the ramp-down shapes the smallest responses.

Results for the entire population of animals used in this study are shown in Figure 12. Each panel of this figure shows the individual results from animals in group 1 (green markers) and group 2 (red markers). The large diamonds with the error bars show the averages ± one standard error. Results show the energy necessary to reach threshold for stimulation (Figure 12A–C), and for the amplitude evoked at 40 μJ/pulse (Figure 12D–F). The averaged values ± their standard errors are listed in Table 1. The ANOVA was not significant.

Figure 12.

Figure 12.

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Optical stimulation accomplished with different pulse shapes. Green markers are for animals with pure tone averages (PTAs) <70 dB SPL and red markers for animals with PTAs >70 dB SPL. Diamond markers with the error bars are the averages ± standard errors. The measurements were conducted at different wavelengths: λ=1,375 nm (A) and (D), λ=1,460 nm (B) and (E), and λ=1,550 nm (C) and (F). Threshold for stimulation has been determined by a visible compound action potential in the recorded traces and is shown in (A), (B), and (C). The maximum amplitude evoked with pulses at 40 μJ/pulse are shown in (D), (E), and (F).

Table 1.

Shown are the average CAP response amplitudes for laser parameters defined in columns 1 and 2.

threshold (μJ/pulse) at 40μJ/pulse (μV)
PTA <70 dB SPL PTA >70 dB SPL PTA <70 dB SPL PTA >70 dB SPL
1375nm square 10.5±2.1 33.9±10.4 126.5±29.8 46.3±8.2
triangle 11.1±4.1 37.3±14.6 104.0±26.6 39.7±9.8
ramp-up 9.1±3.6 34.1±13.5 132.9±30.3 46.6±13.6
ramp-down 21.6±10.1 33.4±10.3 62.9±18.8 37.5±10.2
14460nm square 11.2±2.8 33.4±10.3 103.3±25.2 29.8±8.5
triangle 11.3±3.5 21.4±7.4 83.0±26.1 36.2±5.3
ramp-up 8.9±2.0 23.8±8.0 101.9±30.4 44.1±6.5
ramp-down 20±9.3 27.9±7.9 58.1±18.4 36.2±7.5
1550nm square 12.4±4.8 37.4±12.7 60.8±23.0 30.4±8.8
triangle 13.3±7.7 31.2±11.3 65.2±16.3 13.6±8.7
ramp-up 8.2±2.4 32.0±11.5 89.6±21.5 11.5±7.2
ramp-down 14±3.9 35.8±11.6 37.9±19.8 10.9±6.8
1860nm square 9.5±2.7 41.3±22.2 106.6±36.1 30.0±8.4
ANOVA DF=70 DF=58 DF=71 DF=59
F=0.667 F=0.673 F=1.450 F=2.297
Fc=1.955 Fc=1.995 Fc=1.952 Fc=1.995
P=0.76 P=0.996 P=0.175 P=0.02
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The data are variable and differences are difficult to determine. To reduce the variability and to determine the differences to previously used laser parameters, we have normalized the data to the results obtained with the square shaped pulses at λ=1,860 nm (Figure 9). The radiant energy to reach stimulation threshold was lowest for the triangular and ramp-up pulse shapes at λ=1,460 nm, λ=1,550 nm and for the square pulses at λ=1,860 nm (Figure 9A). The differences are small and statistically not significant. In contrast to the thresholds, ramp-up shaped pulses produced the largest CAP responses when irradiated at 40μJ/pulse (Figure 9B). Again, differences were not statistically significant.

Discussion

The results of this study have demonstrated that INS of the cochlea depends on the radiation wavelength and the pulse shape. The radiation wavelength with the lowest threshold for stimulation was λ=1,460 nm, which differs from the wavelength for the largest CAP response amplitude at either λ=1,460 nm or λ=1375 nm. The effects of changing the radiation wavelength on the response amplitude have been studied in the auditory system before (Baumhoff et al., 2018; Izzo et al., 2007; Kallweit et al., 2016; Schultz et al., 2012b). In the study by Izzo and coworkers, the amplitude of the evoked CAPs increased by changing the radiation wavelength from λ=1,847 to λ=1,873 nm (Izzo et al., 2007). It was the smallest at the shortest optical penetration depth ~322 μm at λ=1,873 nm and saturated at about 700 μm penetration depth at λ=1860 nm. The 378 μm difference between the penetration depth for the smallest amplitude and saturation correlated with the length of the beam path through the spiral ganglion (Izzo et al., 2007). In the present study, for square pulses the largest CAP amplitude was found in the hearing-impaired animals at λ=1,375 nm, the radiation with the longest penetration depth.

In 2012, Schultz et al. (Schultz et al., 2012b) studied the wavelength dependence of the CAP threshold and amplitude with nanosecond laser pulses in the guinea pig auditory system. Since the pulse length was well below 1 μs, the exposure time is shorter than the time for a mechanical disturbance to propagate out of the irradiated volume. This configuration is called stress confinement, typically producing significant pressure waves, which can mechanically stimulate hair cells and produce a CAP (Schultz et al., 2012b). The view that the auditory repose in that study is evoked by a mechanical event is supported by the finding that the response disappears after the deafening of the animals. For those experiments, lower thresholds are found for radiation with the shorter penetration depth in the tissue. In contrast to the experiments in gerbils (Izzo et al., 2007), however, the larger response amplitude was also found for the shorter penetration depths (Schultz et al., 2012b) at λ=1,370 nm, λ=1,550 nm, λ=1,850 nm, and λ=975 nm, which correlates with the higher absorption coefficient of water at these wavelengths. Our results compare for the thresholds but not for the maximum amplitude. The threshold was the lowest for the shortest penetration length of ~322 nm at λ=1460 nm. The largest amplitude was found for the wavelength with longer penetration depths, which would argue for the recruitment of a larger population of neurons for those wavelengths (Izzo et al., 2007). The difference between the results are likely based of the mode of stimulation. While the experiments with the nanosecond pulses (Schultz et al., 2012b) achieved stimulation by a mechanical event, the results in the gerbil (Izzo et al., 2007) and the guinea pig (this study) likely reflect a combined effect of a mechanical event and a direct stimulation of the hair cells or spiral ganglion neurons.

The N1-latency during auditory stimulation with a laser has been examined previously (Schultz et al., 2012b). The N1-latency in normal hearing guinea pigs was about 1.8 ms at CAP-threshold and decreased with increasing stimulus level and increasing N1-amplitude. Similar results were found in our study for the normal hearing animals (Figure 5F). However, animals with compromised cochlear function had longer latencies, close to 3 ms at CAP-threshold. In a paper on the effect of anesthesia and trauma on N1-latencies, it has been shown that threshold elevations can cause the increase of the N1-latency (Brown et al., 1983), which is usually not more than 0.5 ms. The paper also showed that at threshold the N1-latencies depend on the frequency of the stimulus. At 2 kHz, the latencies could be as long as about 2.8 ms. The 3-ms N1 latency reported in the current study does not support a mechanical stimulation of the laser to the low frequency areas of the cochlea, because the latency does not decrease with the increase of the energy level. Rather, it suggests that the response to the infrared neural stimulation (INS) is dominated by an non-acoustic effect, such as a direct interact with the neurons in deaf animals. The fixed latency is likely the result of circumventing the basilar membrane travel time and the synaptic transduction between the inner hair cells and the neurons.

The debate on whether the response in the auditory system to INS originates from a pressure wave or is based on different mechanisms as reviewed in introduction led to another study investigating the pressure generated by laser pulses and its dependence on the wavelength (Kallweit et al., 2016). Their results showed that the pressure measured in a cylinder filled with water depended on the absorption coefficient. The peak pressure amplitude increased with increasing absorption coefficient of water from 0.1 cm−1 to about 57 cm−1. The study also showed that the measured pressure to laser pulses in a fluid tank correlates with the first derivative of the laser pulses and not with their total energy. When stimulating the guinea pig auditory system of hearing animals by placing an optical fiber into the cochlea to directly deliver the radiant energy, the CAP amplitude again correlated with the first derivative of the laser pulses and not with their total energy. If the rise time is an important factor, the pulse shape differences should manifest in pressure differences. The pressure measurements of this study did not show large differences in pressure for different pulse shapes including square, triangle, ramp-up and rampdown pulse shapes with a fixed pulse duration of 100 μs. Rather the energy is an important factor determining the pressure. The experiments in the animals are more efficient for the rampup and triangular pulse shapes. The hearing status of the animals between the two studies differs. While the Kallweit et al. study used hearing animals our study has a mixture of normal hearing guinea pigs and animals with elevated thresholds. In our experiments, the results for the hearing animals appeared to be dominated by an acoustic event, while for hearing impaired animals the results cannot be fully explained by an acoustic event alone and direct interaction of the neuron and the radiation have to be considered.

A different study published in 2018 (Baumhoff et al., 2018) determined the possibility of using INS for acoustic stimulation of the cochlea. In their study, they measured the sound level at the outer ear canal while stimulating with the optical fiber placed in scala tympani of guinea pig cochleae. The pressure they measured compares well to the pressure measured by us in a previous study (Xia et al., 2018) and with the pressure measured in this study. For a 100 μs square pulse, the sound pressure was 55, 57, 60, and 65 dB (re 20 μPa) at 20, 30, 60, and 80 μJ/pulse, respectively. The radiation wavelength was λ=1,860 nm. The pressure values published for the same radiant energies were about 51, 57, 60, and 62 dB (re 20 μPa) (Baumhoff et al., 2018).

Being the pioneer of the field, Wells and coworkers studied in great detail light tissue interactions at the rat sciatic nerve by using the free-electron laser (FEL) at Vanderbilt University. The range of wavelengths suitable for stimulation was explored initially at six wavelengths between λ=2,100 and λ=6,100 nm (Wells et al., 2005b). To estimate the “safest” wavelength for neural stimulation, the ratio of the radiant energy required for tissue (nerve) ablation and for nerve stimulation was determined. It was a factor of about 6 at λ=2,100 and λ=4,000 nm (Wells et al., 2005b). Those wavelengths correspond to minima in the tissue absorption length for the radiation. The authors also argued that minima in the curve for the soft tissue absorption length at wavelengths between λ=300–1,500 nm should not be considered because the longer penetration depth and the increased tissue scattering will reduce the energy at the neurons below the stimulation threshold (Wells et al., 2005b). The results of this study are contrary to this statement. The results of the study have shown for cochlear stimulation that radiation at λ=1,375 nm is similarly efficient to evoke an action potential than radiation at λ=1,860 nm.

Following the initial experiments, more wavelengths have been explored for stimulation as well and different optical sources than the FEL are necessary. At this time, available pulsed laser sources emitting near λ=2,100 nm were the Ho:YAG laser (λ=2,100 nm), the thulium laser (1,910 nm), and the Aculight solid state lasers (λ=1,843–1,877 nm). However, little is known whether radiation between λ=1,260 and λ=1,625 nm is more efficient for INS. This range of wavelengths would be favorable because it is used by the communication industry. Light sources, optical fibers, and polymer waveguides are available for those wavelengths. Moreover, the transmission losses are favorable at those wavelengths. This low-loss radiation range has been divided into five wavelength bands referred to as the O- (λ=1260 to λ=1360 nm), E- (λ=1360 to λ=1460 nm), S- (λ=1460 to λ=1530 nm), C- (λ=1530 to λ=1565 nm), and L-bands (λ=1565 to λ=1625 nm). The fiber loss is lowest for the C- and L-bands, about like 0.2 dB per kilometer (Gasca, 2008). Today, technology has been well advanced for those wavelengths. Waveguides have been developed for those wavelengths, which could be used as alternative systems to deliver the radiant energy into the cochlea. Shorter wavelengths between λ=800 nm and λ=1,300 nm are popular, because the emitters and detectors are less expensive.

Since small light sources became commercially available that emit at wavelengths between λ=1,200 and λ=1,600 nm, this study is to explore whether INS at λ=1,375, λ=1,460, and λ=1,550 nm is possible and whether compound action potentials (CAP) amplitudes evoked from the auditory nerve are comparable to those obtained with λ=1,860 nm. Again, the use of the wavelength λ=1,375 nm is attractive because radiation at this wavelength is used in the communication industry, and the development of optical components, such as semiconductor light sources and waveguides have been optimized, miniaturized, integrated, and are commercially available. Today, emitters for λ=1,375 nm are available off-shelf from SemiNex (Peabody, MA), with the dies’ current-to-light conversion efficiency around 27% at 20°C. Hence, these optical sources are about 2 times more power efficient compared to sources available at λ=1,850 nm. Waveguides for radiation wavelengths between λ=1,200 nm and λ=1,600 nm have also been explored (Matsuura et al., 1999; Matsuura et al., 1994). The development of an optical implant would be more efficient if one could rely on such technology rather than developing novel optical sources. From an engineering point of view, the radiation wavelength at λ=1,375 nm is also advantageous. The conversion efficiency from electrical to optical power, or wall-plug efficiency, is superior for a multi-mode chip at λ=1,375 nm. At 20°C it is ~27% at λ=1,375 nm, about 3 times the wall plug efficiency reported for small optical sources emitting at λ=1,860 nm (10%).

It has been argued that the rate of temperature change is an important factor for INS (Izzo et al., 2007; Kallweit et al., 2016; Plaksin et al., 2017; Rabbitt et al., 2016; Shapiro et al., 2012; Wells et al., 2007a). Therefore, it is plausible that the delivery of the same energy into a smaller volume over the same period of time has a faster temperature change and should be more effective for INS. This notion would also suggest that different pulse rise times would heat a given target volume at a different rate and have been explored previously (Banakis et al., 2011). The effect of pulse shapes and pulse lengths on the radiant energy at λ=1,860 nm required to evoke a CAP in the cochlea has been studied in the guinea pig, gerbil, and cat (Banakis et al., 2011; Izzo et al., 2007). The results showed that the radiant energy had little effect on the CAPamplitudes if the laser pulses were longer than 70 μs. They also showed that at 1860 nm the square pulses were most effective (Banakis et al., 2011) if plotted against peak power. However, if the CAP-amplitude is plotted versus the radiant energy, the ramp-down or square pule shapes are less effective in evoking a CAP than the ramp-up or triangular shapes pulses (Banakis et al., 2011).

Summary

In summary, for neural stimulation with near infrared radiation in the cochlea, sources emitting at λ=1,375 nm or λ=1,460 nm. Furthermore, other waveforms than the square pulse are favorable to deliver the radiant energy. The least favorable pulse shape is the ramp-down. While it has been demonstrated that the ramp-up is typically better or equal to the other waveforms tested, it cannot be ruled out that even more efficient waveforms exist. Modeling efforts are on the way to optimize the waveforms.

Acknowledgements

This work was supported by the NIH, R01-DC011855, R56DC017492 and by the Hugh Knowles Center for Clinical and Basic Science in Hearing and its Disorders at Northwestern University.

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT author statement

Yingyue Xu: Conceptualization; Investigation; Data curation; Formal analysis; Writing - original draft; Writing - review & editing.

Mario Magnuson: Software; Writing - original draft.

Aditi Agarwal: Investigation; Roles/Writing - original draft; Writing - review & editing.

Xiaodong Tan: Investigation; Methodology; Writing - review & editing.review & editing.

Claus-Peter Richter: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Software; Supervision; Validation; Writing -review & editing.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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