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TYPE Original Research
PUBLISHED 08 January 2024
DOI 10.3389/fnins.2023.1288069
OPEN ACCESS
EDITED BY
Jit Muthuswamy,
Arizona State University, United States
REVIEWED BY
Ana G. Hernandez-Reynoso,
The University of Texas at Dallas, United States
Joseph J. Pancrazio,
The University of Texas at Dallas, United States
*CORRESPONDENCE
Yael Hanein
yaelha@tauex.tau.ac.il
RECEIVED 03 September 2023
ACCEPTED 14 December 2023
PUBLISHED 08 January 2024
CITATION
V˙
ebrait˙
e I, Bar-Haim C, David-Pur M and
Hanein Y (2024) Bi-directional electrical
recording and stimulation of the intact retina
with a screen-printed soft probe: a feasibility
study. Front. Neurosci. 17:1288069.
doi: 10.3389/fnins.2023.1288069
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©2024 V˙
ebrait˙
e, Bar-Haim, David-Pur and
Hanein. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is
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the copyright owner(s) are credited and that
the original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
Bi-directional electrical recording
and stimulation of the intact
retina with a screen-printed soft
probe: a feasibility study
Ieva V˙
ebrait˙
e1,2, Chen Bar-Haim1,2, Moshe David-Pur1,2 and
Yael Hanein1,2*
1School of Electrical Engineering, Tel Aviv University, Tel Aviv, Israel, 2Sagol School of Neuroscience, Tel
Aviv University, Tel Aviv, Israel
Introduction: Electrophysiological investigations of intact neural circuits are
challenged by the gentle and complex nature of neural tissues. Bi-directional
electrophysiological interfacing with the retina, in its intact form, is particularly
demanding and currently there is no feasible approach to achieve such
investigations. Here we present a feasibility study of a novel soft multi-electrode
array suitable for bi-directional electrophysiological study of the intact retina.
Methods: Screen-printed soft electrode arrays were developed and tested. The
soft probes were designed to accommodate the curvature of the retina in the eye
and oer an opportunity to study the retina in its intact form.
Results: For the first time, we show both electrical recording and stimulation
capabilities from the intact retina. In particular, we demonstrate the ability to
characterize retina responses to electrical stimulation and reveal stable, direct, and
indirect responses compared with ex-vivo conditions.
Discussion: These results demonstrate the unique performances of the new probe
while also suggesting that intact retinas retain better stability and robustness than
ex-vivo retinas making them more suitable for characterizing retina responses to
electrical stimulation.
KEYWORDS
bi-directional electrophysiology, electrical stimulation, intact retina, soft neural interface,
neurostimulation, neural prosthesis
1 Introduction
Electrophysiological investigations of the retina tap into the electrical activity of the
cells in the retina, particularly the neurons that transmit the signals they receive from
the photoreceptors to the brain. This electrical activity is influenced by various signaling
pathways and reflects on the retina’s developmental stage, circuitry, and viability. The
knowledge gained from electrophysiological interrogation of the retina is important for
understanding underlying mechanisms of retinal function and connectivity. Non-invasive
studies of the electrophysiology of the retina involve techniques such as electroretinography
(ERG), which measures the electrical response of the retina to light stimulation, and pattern
electroretinography (PERG), which assesses the response of the retina to specific visual
patterns (Creel, 2019;Cornish et al., 2021). These tests can be used to evaluate the function
of the retina and to diagnose various retinal disorders, such as macular degeneration and
retinitis pigmentosa, among others (Moschos and Nitoda, 2018;Menghini et al., 2020). ERG
is widely used on animal models in basic and applied research (Vinberg and Kefalov, 2015;
Pasmanter and Petersen-Jones, 2020). Despite their many benefits, non-invasive approaches
suffer from inherently low resolution.
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High-resolution insight into retina electrophysiology can be
gained with ex-vivo retinas (retinas that have been surgically
removed from an animal and maintained in vitro) as a model
system for vision research. There are several benefits regarding
ex-vivo retinas as a model system: Ex-vivo retinas retain their in
vivo organization and structure, including the layers of cells and
their connections, energy metabolism, gradients of electrolytes,
and amino acids (Ames and Nesbett, 1981). This relative integrity
allows the study of the behavior of retinal cells in a physiologically
relevant environment. Ex-vivo retinas can be used to study the
structure, function of the retina, and its response to different
stimuli, such as light, drugs, and electrical stimulation (Field et al.,
2010;Madugula et al., 2022;Shah et al., 2022). Ex-vivo retinas can
be used to study retinal diseases and their effects on retinal function
and structure, thus providing important information needed in the
development of new treatments for retinal degenerative diseases
(Chang et al., 2019;Chenais et al., 2019;Tong et al., 2020). However,
ex-vivo retinas have limitations as a model system. For example,
they may not sustain their full functionality over time, due to
environmental changes and the effects of tissue dissection. High-
resolution strategies to study the electrophysiology of the intact
retina are therefore appealing to explore the differences between the
intact and ex-vivo retinas (V˙
ebrait˙
e and Hanein, 2022).
Although studies on soft neural probes exist, none to the
best of our knowledge, documented bi-directional high-resolution
electrophysiological investigation of the intact retina. Several
studies described flexible electrode arrays that are potentially
suitable for bidirectional retina stimulation and recording,
presenting preliminary testing with ex-vivo retinal model (not
in-vivo) or chronic biostability testing (Rodger et al., 2008;
Montes et al., 2019). Others discussed high-density ultra-flexible
electrode arrays for chronic cortical recordings (Zhao et al.,
2023). Nevertheless, proven high-resolution tools to study the
electrophysiology of the intact retina are presently scarce and only
a few electrophysiological investigations of the intact retina were
described. We refer the interested reader to a mini review on this
specific topic (V˙
ebrait˙
e and Hanein, 2022). In fact, when reviewing
the entire expansive literature on soft neural technologies, it
is apparent that these technologies manifest either high-quality
stimulation or recording (V˙
ebrait˙
e and Hanein, 2021), not both.
Multi-electrode arrays designed for the bi-directional study
of the intact retina demand soft probes with small electrode
dimensions that can record and stimulate the retina at high fidelity.
The probe properties should include bio-compatibility, durability,
and sterilization compatibility. Such retinal probes should not cause
damage to the gentle tissue, and for some applications, optical
clarity is important (V˙
ebrait˙
e and Hanein, 2021;Zheng et al., 2021;
Oldroyd and Malliaras, 2022). Finally, electrode material has to
exhibit stability during stimulation (Schiavone et al., 2020).
In this study, we introduce an innovative method to fulfill
these requisites. The focus of this paper is the development of a
screen-printed soft multi-electrode array for bi-directional neural
interfacing. To showcase the potential of this technology as a neural
interface, we illustrate its application in studying the intact retina.
Flexible high-resolution neural probes are conventionally
realized using photolithography and thin film technology, which
are ideal for high-resolution device fabrication (Herwik et al.,
2009;Boehler et al., 2017). However, under physiological
conditions, these probes tend to suffer from poor stability,
delamination (Boehler et al., 2017), and relatively high rigidity,
imposed by substrate material (e.g., polyimide) and compromised
electrochemical properties. Moreover, soft neuronal electrodes tend
to manifest poor recording performances (V˙
ebrait˙
e and Hanein,
2021). The approach we describe here builds on stacked screen-
printed carbon on soft polyurethane (PU) films. Polyurethane,
as a substrate and passivation material, offers low Young’s
modulus (in the 7–30 MPa range), low water diffusion coefficient
(3.18 ×10−10m2s−1), breathability, compatibility with sterilization
methods, and adherence to ISO 10993-1 toxicity standards
(Rezaei et al., 2010;V˙
ebrait˙
e and Hanein, 2021;Materials and
Technologies, 2022). A screen-printed carbon-based approach
resolves several limitations of photolithography-based thin-film
probes: Carbon electrodes are characterized by high stability,
they are non-Faradaic (within a range allowing safe stimulation),
while also having favorable electrochemical properties to enable
high-resolution recording.
Our previous work in V˙
ebrait˙
e and Hanein (2022)
demonstrated the feasibility of capturing high-quality spontaneous
and light-induced signals from ex-vivo retinal tissue using soft
carbon probes, an improvement over traditional titanium nitride
(TiN) multi-electrode arrays as shown in V˙
ebrait˙
e et al. (2021).
Expanding on this technological foundation, we have taken a step
forward by describing a soft multi-electrode interface with 36
channels, enabling both the recording and stimulation of the intact
retina in a two-way manner. The fabrication process relies on the
layering of two layers of screen-printed carbon, resulting in stable
electrode arrays. To enhance the electrochemical attributes of the
electrodes, we incorporated plasma polymerization of EDOT.
The study also encompasses electrochemical measurements,
which were conducted to characterize the properties of these
electrodes. Furthermore, a feasibility study to showcase the
practical application of these devices for the simultaneous
recording and stimulation of the intact chick retina is presented.
This approach reveals responses within the intact retina, most
notably an enhancement in stability over extended periods
of stimulation and recording. Finally, our study underscores
distinctions between the conditions of an intact retina and
an ex-vivo retina, shedding light on the implications of
each setting.
2 Materials and methods
2.1 Materials
Materials were purchased from Sigma-Aldrich unless specified
otherwise. Phosphate buffered saline (PBS: 2.7 mM KCl, 137 mM
NaCl, 10 mM phosphate buffer, pH 7.4). Artificial cerebrospinal
fluid (aCSF: 5 ×10−3M KCl, 25 ×10−3M NaHCO3, 9 ×10−3
M glucose, 1.2 ×10−3M MgSO4, 1.2 ×10−3M HEPES, 0.5
×10−3M glutamine, 2.5 ×10−3M CaCl2, pH 7.2). Materials
for electrodes include polyurethane film (9,832 F, 3M Medical
Specialties), single-coated polyurethane medical tape (9,832 W,
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3M Medical Specialties), carbon screen print ink (C200, Kayaku
Advanced Materials), 3,4-ethylenedioxythiophene (EDOT).
2.2 Electrode fabrication
Electrodes were fabricated using screen printing of carbon on
polyurethane (PU) films as described previously (V˙
ebrait˙
e et al.,
2021). Screen printing is inherently a low-resolution technology,
thus pre-patterned mesh stencil (Sefar Inc.) properties were
customized to improve printing resolution achieving line widths
as low as 40 µm. Furthermore, to achieve high electrode density
in a small device area (1–4 mm2), layer stacking was employed. The
device consists of three layers: Layer 1 (L1) consisting of PU film (20
µm thick) with screen-printed carbon traces (60 µm in diameter),
Layer 2 (L2) consisting of single-sided adhesive PU film (50 µm
thick) with screen printed carbon traces (60 µm in diameter). L2
also had a laser-cut circular hole (990 µm in diameter) to expose
electrodes in L1, and Layer 3 as a passivation layer consisting of
single-sided adhesive PU film (50 µm thick) with laser-cut circular
holes (1,050 µm to expose electrodes in L1 and 50 µm holes
to expose electrodes in L2). Each layer was prepared separately
then the three layers were aligned and pressed together under a
microscope (Leica M420). In total 36 traces were integrated at 150
µm pitch: 32 recording micro-electrodes (50 µm in diameter), two
additional stimulating electrodes (50 µm in diameter), and two big
reference electrodes.
We refer to these electrodes as “SoftC probe” to reflect the
soft polyurethane substrate, and the conductive material used
(carbon, hence “C”). The total SoftC probe thickness was 160 µm.
A custom-made flexible printed circuit board (PCB) was used to
connect the soft probes with a connector (Omnetics). The binding
between the PCB and the soft array was achieved with a z-axis
adhesive. Detailed schematics of SoftC probe fabrication can be
found in Supplementary Figure 1. In total 10 complete devices
were fabricated and five were characterized (impedance values and
surface area, see details below). Additional test structures were
fabricated for surface and electrochemical analysis.
2.2.1 Screen-printing
Screen-printing of carbon electrodes was achieved using a pre-
patterned mesh stencil. Mesh stencil properties were customized
to improve printing resolution. A mesh (360 wires/inch and
0.0006-inch wire diameter) with 5 µm emulsion over mesh
(EOM) stretched at a 22-degree angle (SEFAR) was used. The
screen was tramp mounted for extra reinforcement and a 70
Durometer squeegee was used to screen print the ink. Printing was
accomplished by a manual application of conductive carbon ink on
a paper-supported PU film. The ink particle size was <10 µm, and
the mesh opening was 56 µm, allowing the ink to pass without
clogging the mesh. The printing step was followed by curing at
130◦C for 10 min.
2.2.2 Laser cutting
A single-sided adhesive PU layer was used to first passivate
Layer 1 and Layer 2 electrodes. A laser cutter (ELAS Ltd.) was
used to define circular holes: 990 and 1,050 µm in diameter in the
center for L1 and L2, respectively, and 50 µm in diameter holes
to expose electrodes in L2. A two-step process was used to prevent
overheating of the adhesive layer: first with a laser intensity of 400
mW to remove the paper support layer, followed by an intensity of
800 mW to remove the remaining two layers (PU and plastic cover).
2.2.3 Plasma polymerized EDOT coating
To complete the electrode fabrication, a plasma polymerized
3,4-ethylenedioxythiophene (ppEDOT) coating was applied. The
process was performed using an RF plasma system (Pico-RF-PC,
Diener electronics), operating at a frequency of 13.56 MHz and a
monomer vapor pressure of 0.1 mbar. A plasma power of 90 W for
15 min was used.
2.3 Surface characterization
Electrode’s approximate diameter and area were estimated and
averaged over five SoftC probes from optical microscopy images
with Image J software. To investigate the surface profile of the films,
environmental scanning electron microscopy (ESEM) and confocal
scanning laser microscopy (CSLM, Lext OLS3000) were used. The
surface roughness was investigated using CSLM, root-mean-square
(RMS) roughness (Rq) and height values were calculated. The
surface wettability was characterized by the water contact angle
using the sessile drop method. The contact angles of 2 µL deionized
water droplets were measured at room temperature (RT) using
a contact angle meter (Rame-Hart model 400). Contact angle
values were calculated using DROPimage Pro software. Contact
angle values were derived from the mean of six droplets deposited
randomly at different locations.
2.4 Electrochemical characterization
The electrochemical properties of test electrode arrays (1,
2, 3, and 4 mm in diameter) were characterized using cyclic
voltammetry (CV) in PBS at scan rates 15, 25, 35, and 45 mV/s
within the −0.5 and 0.5 V range which was estimated to be
within the water window limits. The CV characterization was
done using a three-electrode cell configuration with an Ag/AgCl
reference electrode and a platinum wire as a counter electrode.
CV measurements were conducted using a potentiostat (263A
Princeton Applied Research) and recorded using the PowerCV
software (Princeton Applied Research). The cathodal and anodal
charge storage capacities (CSC) were calculated using the time
integral of the cathodal and anodal current over a potential range
defined as the limit for water electrolysis window derived from CV
performed at a scan rate of 45 mV/s. The same setup was used to
test charge injection for stimulating electrodes, with cathodic-first
symmetric biphasic current pulses. The applied pulse width was 50
ms and the inter-pulse delay was 10 ms.
The impedance of the SoftC probe electrode (∼50 µm in
diameter) was measured at frequencies ranging between 25 and
5,000 Hz in aCSF using RHD2132 recording head stage and an
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RHD 2000 USB interface board (Intan Technologies LLC, Los
Angeles, CA, USA). Electrophysiological data in this study was
recorded with a single device in which we monitored stable
impedance (274 kat 1 kHz) for 18 recording channels over 120
days. The other 14 channels had higher impedance values (in the
range of 10–30 M).
Voltage transients generated by stimulating an electrode of
SoftC probe were measured in an aCSF solution. Voltage transient
recording was done 50 µm above the electrode surface using
a glass capillary electrode filled with 3M KCl, mounted on a
computer monitorized micromanipulator (PatchStar, Scientifica)
vs. an Ag/AgCl reference electrode in aCSF. The measuring
unit consisted of a voltage amplifier (ELC-03XS, npi electronic
GmbH). A charge-balanced biphasic current pulse of 400 µs,
with increasing amplitudes, was injected to a single stimulating
electrode of SoftC probe, using an external stimulator (STG4002,
MultiChannel Systems).
2.5 Animal care and use
All experimental procedures were performed in accordance
with Animal Welfare Law—Experiments in Animals 1994 and
under approval by the Institutional Animal Care and Use
Committee at Tel Aviv University (permit number: TAU-MD-
IL-2207-173-1). Fertilized chick eggs were incubated at 37◦C,
until embryonic day 14 (E14) followed by rapid egg opening and
embryo decapitation. The enucleation and eye preparations were
performed in oxygenated (95% O2, 5% CO2) chick aCSF solution
under a binocular microscope. Cornea, lens, sclera, and vitreous
humor were removed and for ex vivo experiments the retina was
detached from the pigmented epithelium, dissected into 4 ×4
mm squares, and laid on the MEAs with the ganglion cell layer
(GCL) facing down. To improve coupling between the tissue and
the electrodes, a polyester membrane filter (5 µm pores, Sterlitech)
and a stainless-steel washer were placed on top of the retina. For
the experiments in the eye, the eye cup was carefully transferred
to the custom-made holder where the cornea, lens, sclera, vitreous
humor, and SoftC probe were gently laid on the surface of the
retina. During experiments, the retinas were kept at physiological
conditions, at a temperature of 34◦C, and perfused (2–5 mL/min)
with oxygenated (95% O2, 5% CO2) chick aCSF solution. In total,
five retinas were used for each preparation (ex-vivo and intact).
2.6 Electrophysiolgy
To reduce the potential for failed experiments, we used two
well-characterized devices (softC and TiN MEA) to perform the
electrophysiological study. Throughout the entire duration of the
study, we monitored the softC device’s stability and consistent
performance. Impedance measurements were performed before
each experiment, following the experiment, the device was
thoroughly cleaned with deionized water (DI) and stored. We did
not encounter any significant deviations in electrode impedance
values or noise levels that could have affected the reliability of
our data. Coupling between the tissue and the electrodes was
established by gently laying the probe on the surface of the
retina, using a manual micromanipulator (Kite-R, World Precision
Instruments). The retinas were kept at physiological conditions, at a
temperature of 34◦C, and perfused (2–5 mL/min) with oxygenated
chick aCSF solution. Neuronal signals were amplified and acquired
at 25 kHz per channel, with an RHD2132 recording head stage and
an RHD2000 USB interface board (Intan Technologies). Retinal
stimulation was carried out by injecting a biphasic pulse of 300 µs
with an inter-phase delay of 60 µs to a single electrode of the SoftC
probe, using electrical stimulus generator (STG4002; MultiChannel
Systems). Stimulation amplitudes used in the study varied between
1 and 100 µA at either 1 or 10 Hz stimulation frequency.
Intact retina recordings and stimulation were compared to the
ex-vivo retina placed on a 30 µm diameter TiN electrode MEAs
(MultiChannel Systems)—ex-vivo retina model. Ex-vivo neuronal
signals were amplified with a MEA1060-up amplifier (gain ×1,100,
MultiChannel Systems), digitalized using a 64-channel analog to
digital converter (MC_Card, MultiChannel Systems), and recorded
(MC_Rack, MultiChannel Systems).
The recorded signals were analyzed offline in DataView
(software by Heitler W J, version 11.11.1, University of St Andrews,
Scotland, UK). Raw traces were first filtered to remove local DC
by subtracting a local moving average from the signal, then the
signals were filtered with 300–3,000 Hz (Butterworth 2nd order).
The stimulation artifact was removed by identifying stimulus onset
and interpolating 2.5 or 5 ms before and after the stimulus artifact
event for direct and indirect response visualization, respectively.
Retinal spikes were detected using threshold-crossing criteria [five
times the standard deviation (SD) of noise]. Recorded spikes
were processed into individual units using a principal component
analysis. Automatic clustering was performed using a built-in
unsupervised algorithm by Bouman (1997). Signal-to-noise ratio
(SNR) was evaluated for ex-vivo and intact retina recordings during
10 Hz stimulation. RMS noise was calculated 80 ms pre-stimulation
and RMS signal was calculated in between the stimulation pulses
at six random time points (Supplementary Table 1). Intact retina
responses to electrical stimulation from all recorded channels is
additionally presented in Supplementary Figures 7,8. Zoom in view
to retina responses (both ex-vivo and intact) during 10 Hz electrical
stimulation is presented in Supplementary Figure 9.
3 Results
Soft electrode arrays (SoftC) were realized using screen printing
of carbon ink on soft and thin polyurethane (PU) films. Given
the relatively low resolution of screen printing, stacking multiple
layers was used to achieve a high electrode count (Figure 1a).
Previously, we presented arrays with only eight electrodes (V˙
ebrait˙
e
and Hanein, 2022). In this study, we increased the electrode number
to 36 electrodes. We reduced the electrode diameter from 80 to
50 µm and inter-channel distance from 430 to 150 µm, achieving
higher spatial resolution and electrode count (Figure 1a). The
overall dimensions of the soft array probe are 6 mm ×2.6 cm ×160
µm (width, length, total thickness, respectively) and effective array
area of ∼1.13 mm2incorporating 32 recording-stimulating micro-
electrodes, two additional stimulating electrodes, and two big
reference electrodes. The final SoftC probe is shown in Figure 1b,
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FIGURE 1
36-Channel SoftC probe. (a) The layout of the SoftC probe consists of two layers with printed carbon traces and a top passivation layer. (b) Picture of
a fabricated complete SoftC probe. Traces are connected to the custom-made flexible PCB and Intan high-density connector. Zoom-in image of the
SoftC probe. The top zoom-in image shows four electrodes: two channels (orange circles) of layer 1 and 2 channels (red circles) of layer 2. Red circle
diameter 50 µm. (c) ESEM images of carbon on PU substrate top (left) and cross-section (right) views. Insets: magnified views. (d) Picture showing
experimental set-up: chick embryo eye placed in a custom printed holder-chamber, SoftC probe.
consisting of a soft part (with the carbon electrodes), a flexible
PCB, and a high-density connector. The electrodes in Layer 2
have a circular shape, as defined by the laser cut hole, and
electrodes in Layer 1 have a slightly deformed shape (Figure 1b
inset). Electrode’s approximate diameter and area were estimated
and averaged over five SoftC probes from optical microscopy
images (Supplementary Figure 2B), resulting in an overall average
diameter of 58.9 µm (SD = 10.7) and an area of 1,475 µm2
(SD = 349). Environmental scanning electron microscope (ESEM)
imaging was used to examine the electrode surface. Figure 1c shows
ESEM images depicting a rough carbon surface, which is important
for enhancing an effective electrode surface area. Additionally, a
confocal scanning laser microscopy of the carbon on PU film
(Supplementary Figure 4) was used to derive a surface roughness
of ∼1µm and a height of ∼11 µm. Contact angle measurements of
the electrode surface (Supplementary Figure 5) demonstrate angles
of 32.5 (SD = 2.4) and 81 (SD = 4) for ppEDOT-coated and
bare electrodes, respectively. A complete device introduced into an
enucleated chick eye is shown in Figure 1d.
Electrochemical measurements were performed to validate
electrode performances, including non-Faradaic behavior, specific
DC capacitance, impedance, charge injection, and stability
(Figure 2). Cyclic voltammetry (CV) of screen-printed electrodes
was first used to validate the non-Faradaic nature of the electrodes
and to characterize the water window. The low-frequency
capacitance of the electrodes was also extracted (Figures 2A,B).
In particular, CV measurements reveal the effect of ppEDOT
coating. Screen-printed electrodes coated with ppEDOT obtained a
specific capacitance of 0.92 mF/cm2(compared with 0.09 mF/cm2
for bare electrodes), averaged over five samples each. The high
specific capacitance values are important for low-noise electrical
recordings at small electrode dimensions. Figure 2A shows that the
boundaries of the electrochemical potential window for ppEDOT
coated electrode in PBS are −0.5 and +0.5 V (Figure 2A). CV
measurements also reveal a capacitive behavior and water window
values typical to carbon/ppEDOT electrodes. The cathodal and
anodal charge storage capacities (CSC) were calculated by the
time integral of the cathodal and anodal current over a potential
range determined by a water electrolysis window. Values are
presented in the Supplementary Figure 3 for both electrodes. CSC
was evaluated to be 2.7 and 2.0 mC/cm2for coated and uncoated
carbon electrodes, respectively.
Charge transfer tests were carried out to categorize the
stimulating electrodes in terms of their reversibility and maximum
charge injection capacity (Figure 2C). The charge injected in the
first half-phase is seen to be immediately compensated for by
reversing the polarity over the second half. A charge injection
test with a specific capacitance of 0.12 mC/cm2(75 µA, 50 ms)
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FIGURE 2
SoftC probe characterization. (A–C) Electrochemical characterization was performed on test structures with electrodes 1, 2, 3, and 4 mm in
diameter. (A) Cyclic voltammetry scan of ppEDOT uncoated and coated electrode (D = 1 mm) under 45 mV/s scan rate. The inset shows the
charging current vs. the scan rate of electrodes, dashed lines are linear fits. (B) Electrode capacitance vs. electrode area for ppEDOT coated and
uncoated electrodes measured under 45 mV/s scan rate. (C) Charge injection test with cathodic-first symmetric biphasic 50 ms current pulses. (D–F)
Voltage transient measurement 50 µm above SoftC probe stimulating electrode with a glass-pulled capillary electrode (D) in response to
charge-balanced biphasic current pulse (400 µs with 100 µs interpulse delay, e inset) at increasing current amplitudes. (E) Transient voltages, Vt,
measured in response to biphasic current pulse. (F) The cathodic peak of the Vts as a function of the injected current. The equivalent charge density
is shown above. (G, H) SoftC probe electrode impedance measurements using Intan system in aCSF. (G) Impedance magnitude at 1 kHz of all 32
electrodes over time. (H) Electrode impedance and phase vs. frequency (25–5,000 Hz). The mean and standard deviation of the impedance of 18
electrodes (channels: 2–11, 13–15, 17, 19–20, 29, 31) were measured on day 66.
shows a potential increase in the 200 mV range at the electrode
interface.
To validate the stability of the electrode under electrical
stimulation, the potential above the electrode was also measured
using a glass-pulled capillary electrode mounted on a motorized
stage. Voltage transient Vts values at different charge densities
show an almost linear increase through the 0–2 mC/cm2range.
The values of the cathodic peak are similar to those measured at
the same distance with commercial 30 µm diameter TiN MEAs
(Supplementary Figure 6). In a prior study, screen-printed carbon
electrodes on polyurethane films were shown to be compliant
with EtOH and ultraviolet (UV) sterilization methods as well as
stable in aCSF solution (at 40◦C) for over 6 months with no
delamination or change in the resistance values (V˙
ebrait˙
e et al.,
2021). Here, the impedance stability of the electrodes in the
aCSF solution was validated (Figure 2G) over 120 days. Impedance
measurements at the 0.025–5 kHz range on day 66 showed stable
values (274 kat 1 kHz).
Electrical activity in the intact retina in response to electrical
stimulation was studied and is presented in Figure 3. A developing
chick retina at embryonic day 14 (E14) was used as a blind
retina model for these studies (N= 5 retinas). Chick retina
at E10–E18 has developed and functional ganglion cell layer as
well as inner and outer layers but an undeveloped photoreceptor
layer with no responses to light until E19 (Mey and Thanos, 2000;
Sernagor et al., 2001). The soft probe was placed inside an open
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FIGURE 3
Intact retina recording and stimulation with SoftC probe. (A) Picture of 36-channel SoftC probe placed inside the enucleated eye on the intact retina.
(B–E) Electrical recordings by SoftC probe during a delivery of 50 µA 300 µs charge-balanced biphasic 100 pulse train stimulation at 10 Hz frequency
(Retina #1). (B) Ch8 and Ch20 impedance amplitude in aCSF before the experiment and after placement on the retina show an increase of 25 and
46% from the initial value at 1 kHz, respectively. (C) Second order Butterworth 300–3,000 Hz filtered trace (ch20) before artifact removal. Red vertical
lines indicate stimulation onset. Zoomed view to six pulses. (D) Electrical recordings of 16 channels over the whole stimulation train after artifact
removal (−4 and +4 ms were set to 0 mV at the onset of stimulus). Zoomed view to three dierent time points of stimulation train of ch8, ch15, and
ch20. (E) Spiking waveforms detected during the stimulation period. Spike shape classification by principal component analysis (PCA) resulted in 3, 2,
and 2 spike waveforms for channels 8, 15, and 20, respectively. Grey lines—10 to 50 spikes superimposed, black line—average waveform. (F) Artifact
removed (non-filtered trace) short latency responses during 20 µA 300 µs charge-balanced biphasic stimulation at 1 Hz frequency (Retina #2).
blind chick eye (E14) mounted on a custom-made holder
(Figure 3A). The eye was kept submerged in oxygenated aCSF (for
as long as 3 h).
Electrode impedance measurements were first performed in
aCSF and after placement against the retina to validate tissue-
electrode coupling (Spira and Hai, 2013;Majdi et al., 2014).
The measured impedance increased or remained the same. In
six channels (out of 16 measured) impedance values (at 1 kHz)
increased by 4–50% from the baseline value measured in aCSF
(Figure 3B), indicating coupling. Bi-directional measurements
were then used to study retina responses to electrical stimulation.
For all SoftC probe stimulations of the intact retina, 300 µs
charge balance biphasic pulses were used at either 1 or 10 Hz
frequency. Figure 3C illustrates recorded traces contaminated with
stimulation artifact, thus it was removed during offline analysis.
The filtered and artifact-removed (see Section 2) data reveal a clear
retina response to electrical stimulation. Both direct (Figure 3F)
and indirect activation (Figures 3C–E) were observed. The first
is typified by short, high-amplitude responses that increase with
increasing current amplitude. Indirect activation is characterized
by low-amplitude and long latency responses. We note that retina
responses were clearly visible in those channels with improved
coupling (e.g., channels 15, 20, and 8 with 6, 25, and 46% increase
in impedance, respectively). These results suggest that despite the
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FIGURE 4
Spontaneous and stimulated retina activity. (A) Responses to electrical stimulation in ex-vivo and intact retina to 1.69 and 0.76 mC/cm2stimulation,
respectively. A typical spontaneous wave in ex-vivo retina recordings (left, zoom in inset). No such activity was observed in intact retinas (right). Insets
(i-ii) superimposed direct responses. (B) Stimulus-response (raster plots) to 10 Hz stimulation over 10 s in the ex-vivo retina (left; 0.68 and 1.49
mC/cm2stimulation for top and bottom, respectively ) and in the intact retina (right; 0.76 mC/cm2stimulation). Each row corresponds to 100
consecutive stimulation pulses. Red arrowheads indicate stimulus onset.
curved nature of the intact retina, the soft probe achieved good
electrical coupling, especially in those electrodes with high signal-
to-noise ratio responses.
Finally, we compare the indirect responses (long latency) in the
intact retina with ex-vivo conditions. In the intact retina, responses
appear to remain stable upon repetitive stimulation lasting for 10
s (Figures 3D,4A,B). Similar stimulation in ex-vivo conditions is
typified by clear desensitization, especially with higher stimulation
frequencies (Jensen and Rizzo, 2007;Chenais et al., 2021;Li
et al., 2022), and a strong sensitivity to spontaneous activity,
which completely abolish the response to stimulation. Figure 4A
presents an example of a spontaneous wave direct inhibition of
retina responses to electrical stimulation in the ex-vivo retina,
whereas in the intact retina, such an effect was not detected. We
observed almost no spontaneous activity [Figure 4A (right)] in the
intact retina experiments (N= 5 retinas). This is, to the best of
our knowledge, the first direct electrophysiological evidence for a
discrepancy between ex-vivo and intact retina conditions.
4 Discussion
Soft electrode arrays, engineered for bi-directional electrical
interfacing with the retina, were realized and put through testing.
The probes were realized by stacking two screen-printed soft layers.
We were able to increase the electrode count and realize electrode
arrays with good recording and stimulation performances, even
when positioned against the intact retina. Various flexible and
soft probes were investigated in the past. We briefly discuss
a few representative examples. In Graudejus et al. (2009), 11
recording electrodes were realized using photolithography and
electrodeposition on 280 µm thick layer of an elastomeric
silicone substrate. Silicon nanomembrane transistor arrays on
polyimide (few µm thick, Young’s modulus 1.3–3 GPa) were
used to form dense arrays of thousands of amplified and
multiplexed sensors (Viventi et al., 2011). The use of a grid
structure was also used to help reduce the rigidity of a recording
device as demonstrated in Reference (Khodagholy et al., 2015).
These and many other approaches have not demonstrated high-
density recording and stimulation with thin and soft support.
Transitioning from rigid or flexible substrates to soft ones usually
comes with a cost in performance. For example, CNT micro-
electrodes (30 µm in diameter) on Si/SiO2, can reach high
specific capacitance in the range of 3–10 mF/cm2(Gabay et al.,
2007). Values obtained with state-of-the-art Titanium Nitride
(TiN) on glass are in the 2 mF/cm2(Gabay et al., 2007).
CNT micro-electrodes on soft support will typically have a
specific capacitance in the range of 1–2 mF/cm2. ECoG probe
using CNTs on PDMS electrodes exhibits specific capacitance
of 1.5 mF/cm2, low impedance values of approximately 30 k
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at 1 kHz, and charge storage capacity of 0.35 mC/cm2(Yang
et al., 2022). Faradaic silver nanoparticles/PEDOT:PSS [Poly(3,4-
ethylenedioxythiophene) Polystyrene Sulfonate] electrodes on
polyimide can reach a very low impedance value of 200
(at 1 kHz), exhibit capacitance of 0.4–60.6 mF/cm2and charge
storage capacity of 1.083–2.8 C/cm2by increasing PEDOT:PSS
coating from 1 to 10 layers (Almaris et al., 2020). The soft and
thin electrode arrays we presented here achieve good recording
and stimulation performances despite their very simple and
straightforward fabrication process and as such represent an
advantage compared with alternative approaches.
These pliable probes are particularly adapted to conform
to the curvature of the eye’s retina, providing a distinctive
chance to investigate the retina in its undisturbed state. Using
the SoftC technology, we demonstrated, for the first time,
electrophysiological differences associated with retina conditions
(i.e., ex-vivo vs. intact retina). Differences associated with
spontaneous activity were observed thanks to the ability to
record directly from the intact retina. Differences associated with
evoked responses were facilitated by the bi-directional ability
presented here.
Discrepancies between in vitro,ex-vivo, and intact tissues
are well documented (Alaylio˘
glu et al., 2020). Ex-vivo retinas,
in particular, are expected to show some structural damage and
decreased functional responses compared to intact retinas, owing
to mechanical manipulation and the loss of optimal biochemical
support. These differences imply that the ex-vivo preparation may
not accurately reflect the in vivo state of the retina. Among various
expected effects, these differences are expected to profoundly affect
retina electrophysiology.
One such effect is spontaneous waves. Retina spontaneous
activity is a well-established phenomenon of developing and
degenerating retinas and is known to interfere with retina
stimulation strategies (Wong et al., 1998;Goo et al., 2016;Haselier
et al., 2017). In a previous study (V˙
ebrait˙
e et al., 2021), we
presented bio-potential measurements from intact retinas. In
those measurements, spontaneous activity was observed in only
2 out of 11 intact eyes. On those rare appearances, the recorded
spontaneous activity was shorter in duration and had a low signal-
to-noise ratio compared to that typically observed ex-vivo. In our
previous study, the SoftC probes did not have electrical stimulation
capability, and the observation of spontaneous retinal activity was
an indicator of good probe-retina interface and retina viability. In
this study, we report almost no spontaneous activity in the intact
retina (N= 5 retinas). Importantly, in the present study, the ability
to electrically stimulate retina responses serves as a powerful tool
to validate retina viability and to provide greater confidence in
asserting the lack of spontaneous activity in intact retina.
Studying retina responses to electrical stimulation is pivotal in
the domain of artificial vision (also known as retinal prosthesis
or a bionic eye). Electrical stimulation works by using electrodes
implanted in the retina to stimulate remaining healthy cells
and transmit visual information to the brain (Bloch et al.,
2019;Ayton et al., 2020). For individuals with severe vision
loss, electrical stimulation of the retina can provide a means of
regaining functional vision, allowing them to perform basic daily
activities, such as recognizing faces, reading, and navigating the
environment (Castaldi et al., 2016;Hallum and Dakin, 2021).
Electrical stimulation of the retina has shown promising potential
in treating certain forms of visual impairments, particularly
retinal degenerative diseases such as retinitis pigmentosa and age-
related macular degeneration (Chow et al., 2002;Palanker et al.,
2022). There are several lingering challenges related to electrical
stimulation of the retina, primarily: Stability, efficacy, safety, and
feedback (Ryu et al., 2019). Having demonstrated the intact retina
responses to electrical stimulation, it is interesting to discuss these
results focusing on stability, efficacy, and safety in comparison to
the extensively published ex-vivo retina data.
In this investigation, we were able to observe both direct and
indirect responses in the intact retina. Interestingly, the responses
observed in the intact retina remain stable, even after extensive
stimulation (Figures 3D,4B). Similar stimulation in ex-vivo retina
is typified by clear saturation (Chenais et al., 2021). We hypothesize
that the origin of this interesting discrepancy is the physiological
state of the retina, which gives rise to better stability of the intact
retina under what appears to be better conditions. In the realm of
retina stimulation, high efficacy stands for the ability to achieve
local stimulation with high temporal resolution. We expect that
ganglion and bipolar cell response to stimulation, and in particular,
its efficacy will be affected by whether retina circuits are intact
or have lost their natural support. These issues will be further
discussed and analyzed in a separate publication.
A major concern in electrical neural stimulation is safety.
Moreover, the current or charge delivered has to be minimal.
Identifying optimal parameters for high-fidelity cell activation is
still an active research question (Colodetti et al., 2007;Madugula
et al., 2022). Retina activation (direct or indirect) effectiveness
is influenced by stimulation parameters such as waveform shape,
amplitude, duration, frequency, and polarity (Tong et al., 2020).
For example, some studies point out the advantages of monophasic
stimulation while others suggest that biphasic stimulation shows a
lower activation threshold and shorter response latency. Generally,
biphasic stimulation is widely used for retinal implants (Jensen
and Rizzo, 2009;Boinagrov et al., 2014;Jalligampala et al., 2017;
Celik and Karagoz, 2018;Meng et al., 2018). Another safety
concern is oxidative stress. Overproduction of reactive oxygen
species (ROS) is known to affect the electrode-tissue interface,
which can lead to foreign body response, damage to the cells
and tissue, and can also create structural damage, and changes in
properties of the implanted device (Takmakov et al., 2015;Ereifej
et al., 2018). The retina is particularly sensitive to ROS, which
can lead to a range of retinal disorders in vivo, including age-
related macular degeneration (AMD), diabetic retinopathy, and
retinitis pigmentosa, among others (Wang et al., 2022). Thus,
the retina exhibits several protective mechanisms, including the
presence of antioxidants such as glutathione, vitamins, and taurine
as well as enzymes such as superoxide dismutase and catalase.
Additionally, the retina’s high metabolic rate and high oxygen
consumption are regulated to minimize the production of ROS
(Léveillard and Sahel, 2017;Domènech and Marfany, 2020). It
is therefore not too surprising that extensive stimulation ex-vivo
could lead to various instabilities, some of which may be associated
with the electro-chemistry at the electrode interface unless
electrode passivation is explicitly used (Fromherz and Stett, 1995;
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Eickenscheidt et al., 2012). How more efficient stability is offered by
in-vivo conditions is an important topic for further investigations.
An additional clear bottleneck in the field of retina stimulation
is the lack of informative feedback on the efficacy of a specific
stimulation paradigm. Optimized stimulation parameters (e.g.,
pulse duration, shape, and rate) are important to reduce threshold
and energy, provide more selective stimulation (ganglion vs.
bipolar cells), reduce fading, and increase localization. In pre-
clinical stages, researchers have to rely on animal behavior, evoked
potential responses, or data collected from dissociated retinas.
These parameters provide only indirect information. For example,
the selectivity between ganglion cell, fiber layer, and bipolar cell
stimulation has received much attention (Tong et al., 2020), as it
is hypothesized that axon fiber activation contributes to reduced
localization. Moreover, reports on optimal stimulation parameters
are contradictory. Some studies suggested that particularly long
(10 ms) pulses can lead to selective activation of bipolar cells
(Boinagrov et al., 2014). While Tong et al. (2019), showed that
long pulses do not lead to the desired selectivity. One possible
explanation for the discrepancy between reports is electrode
geometry. An alternative explanation is the different physiological
conditions of the retina. Exploring this effect in the intact retina
will help in understanding the role of retina physiology in
retina stimulation. Desensitization, owing to rapid stimulation, is
another major unresolved issue in neural stimulation. In particular,
the effect of pulse trains on ganglion cells vs. bipolar cells.
The technique reported here can help alleviate the uncertainty
associated with electrical stimulation of the retina and will be used
in future explorations.
The probes presented in this study can benefit a wide range
of neurotechnological applications, far and beyond the study of
the retina. Despite the fabrication simplicity, the arrays are stable
while also manifesting high-quality performances for bi-directional
electrical interfacing. This can be handy in the realm of cortical
electrodes, subdermal implants, and much more.
Despite its numerous advantages, the technology presented in
its current form is not without drawbacks. One notable limitation
is the yield of the manual printing and layer stacking procedures.
An additional challenge is the probe thickness and lack of
permeability. Enhancements in probe design, including reducing
the thickness of the device, incorporating perforations to
increase oxygenation, and maintaining uniformity throughout the
fabrication process, coupled with a more extensive validation
process both in laboratory settings and within living organisms, will
provide stronger support for establishing the showcased technology
as a resilient instrument in upcoming research endeavors.
To summarize, this feasibility study demonstrates a novel
approach featuring a high-resolution probe for electrophysiological
investigation of the intact retina. We conducted electrical
stimulation while simultaneously recording both direct and
indirect responses with the same device. Our findings revealed
low levels of spontaneous activity and stable responses under
high-frequency stimulation compared to ex-vivo retinas. These
initial observations suggest distinctions between ex-vivo and intact
retina conditions. However, further investigations are warranted to
comprehensively explore the underlying mechanisms responsible
for these differences.
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Institutional Animal
Care and Use Committee at Tel Aviv University. The study
was conducted in accordance with the local legislation and
institutional requirements.
Author contributions
IV: Conceptualization, Investigation, Writing—original draft,
Writing—review & editing. CB-H: Investigation, Writing—review
& editing. MD-P: Investigation, Conceptualization, Writing—
review & editing. YH: Conceptualization, Funding acquisition,
Supervision, Writing—original draft, Writing—review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This project
was supported by an Israel Science Foundation (ISF) Grant
(No. 538/22) and the European Research Council (ERC) grant
(Outer-Ret—101053186).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The author(s) declared that they were an editorial board
member of Frontiers, at the time of submission. This had no impact
on the peer review process and the final decision.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnins.2023.
1288069/full#supplementary-material
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