A flexible photoacoustic retinal prosthesis

Domain: chemistry
Published: 2025-12-23
Editorial outcome: minor_revision

Abstract
Retinal degenerative diseases of photoreceptors are a leading cause of blindness with no effective treatment. Retinal prostheses aim to restore sight by stimulating residual retinal cells. Here, we present a photoacoustic retinal stimulation technology. We designed a polydimethylsiloxane and carbon-based flexible film that converts near-infrared laser pulses into a localized acoustic field with 51-µm lateral resolution, allowing precise stimulation of mechanosensitive retinal cells. This photoacoustic stimulation robustly and locally modulated retinal ganglion cell activity in both wild-type and degenerated ex vivo rat retinae. In animals subretinally implanted with a millimeter-sized photoacoustic film, pulsed laser stimulation generated neural modulation along the visual pathway to the superior colliculus, as measured by functional ultrasound imaging. The biosafety of the film was confirmed by the absence of short-term adverse effects, while local thermal increases were measured below 1°C. These findings demonstrate the potential of photoacoustic stimulation for high-acuity visual restoration in blind patients.

Introduction
Retinitis pigmentosa and age-related macular degeneration affect millions of people worldwide1, resulting in irreversible photoreceptor degeneration and blindness. Currently, there is no effective drug treatment for preventing photoreceptor loss. Retinal prostheses are implantable devices designed to stimulate the residual retinal layers to restore vision2. To date, two retinal prosthesis designs, which both primarily use electrostimulation to restore vision, have been approved for commercial implantation3. However, both were later removed from the market, facing challenges such as poor spatial resolution and small restored visual field. The more recent photovoltaic-based PRIMA implant, one of the most advanced retinal prostheses currently in clinical trials (NCT04676854), offers a spatial resolution of 100 µm, a median visual acuity below the threshold for legal blindness4, and a limited restored visual field of 7°.

Focused ultrasound is a promising noninvasive approach for retinal stimulation5. It has been shown to evoke stable responses in the ex vivo salamander retina with a 90-µm lateral resolution at 43 MHz6 and in the in vivo blind RCS rat retina with a lateral resolution of around 400 µm with a 4.5-MHz transducer 2D-array7–9. However, it still faces safety challenges to meet FDA safety threshold for ophthalmological use7.

Photoacoustic modulation is an emerging method for high-precision neural stimulation10,11. A localized ultrasound field is generated by the photoacoustic agents upon excitation with a pulsed laser, and used to stimulate neurons. High spatial resolution stimulation of single neurons has been demonstrated through a fiber-based photoacoustic emitter10.

In this study, we investigated photoacoustic retinal stimulation as an alternative strategy for restoring vision. We here provide evidence of its efficacy both ex vivo and in vivo on healthy and degenerated retinae, as well evidence of the biosafety of the photoacoustic film in short-term implantation.

Results
Fabrication and characterization of the flexible photoacoustic film
The working principle of photoacoustic retinal stimulation is illustrated in Fig. 1a. A 1030-nm pulsed laser is delivered to the back of the eye onto the retina, illuminating the subretinally-implanted flexible photoacoustic (PA) film. Laser absorption by the film produces transient heating, which causes thermal expansion and compression of the material, thereby generating pulsed ultrasound waves. The generated ultrasound waves stimulate the mechanosensitive retinal cells6,8,12, resulting in a change in retinal ganglion cell activity, which is then transferred to the brain.

The PA film (Fig. 1b) consists of candle soot (CS) as the absorber material, sandwiched between two layers of polydimethylsiloxane (PDMS), which acts as the thermal expansion material. The film has a Young’s modulus of 2.12±0.10 MPa to minimize the immune response13,14 once implanted (Supplementary Fig. S1). Upon excitation with 4.2-ns laser pulses at 7 µJ per pulse, the PDMS/CS/PDMS film emitted ultrasound pulses with a peak-to-peak pressure of 146.2 kPa measured 0.9 mm away (Fig. 1c). At the surface of the film, the conversion efficiency is estimated at 26 kPa.µJ-1 based on the distance-dependent pressure profile measured (Supplementary Fig. S2). PA signals were found to have a central frequency of 42.2 MHz and −6 dB bandwidth ranging from 29.6 to 59.9 MHz. This central frequency is an intrinsic property of the film, since the ultrasound frequency of a photoacoustic device is determined by the effective absorption depth of light. The flame synthesis process used to fabricate our CS-based film does not allow concentration tuning for modifying the ultrasound frequency. This central frequency has been demonstrated to activate ex vivo salamander retinae with a lower intensity threshold compared to lower acoustic frequencies15. These data suggest that the PDMS/CS/PDMS film is a promising photoacoustic converter for retinal stimulation.

The spatial distribution of the ultrasound field generated by the PDMS/CS/PDMS film was further mapped by PA-field microscopy (Fig. 1d, Supplementary Fig. S7, Fig. 1a from Chen et al.16). A 50-µm optical fiber was attached to the PA film to ensure a 50-µm-diameter illumination area. The axial pressure profile shows that the maximum acoustic signal is generated at the Z = 34 µm from the surface of the film upon illumination and attenuates to 50% of its peak value at Z = 232 µm (Fig. 1d, right). The lateral width (W) of the acoustic field, quantified by the full width at half maximum, measures W = 51 µm at Z = 34 µm and increases with axial depth to W = 93 124 µm at Z = 100 µm (Fig. 1e). These results confirm that under a confined illumination, the PA film produces a highly localized, 51-µm lateral ultrasound field comparable to the size of illumination, opening up potential for retinal stimulation with sub-100-µm resolution.

Measured acoustic pressure exhibited a linear relation with the incident laser energy per pulse (Fig. 1f), which indicates that the output pressure can be precisely modulated by adjusting the input laser energy.

The greatest heat increase is assumed to be generated at the level of the PA film, as a consequence of the laser light absorption. Ultrasound absorption could cause an additional increase in temperature, but it is expected to be much lower due to the very low acoustic energy. To ensure that the laser light absorption by the designed PA film is not associated with a substantial and detrimental temperature increase, we measured the temperature at the surface of the PA film. The tested laser conditions were consistent with those employed in the following ex vivo retinal stimulation experiments in terms of pulse energy and repetition rate, but the pulse duration (50 ms) was longer than the maximum duration (30 ms) used in the next section. We observed a maximum temperature rise of 0.52±0.09 °C (Fig. 1g). The baseline change due to cumulative thermal effects was 0.21 °C after 40 s. This value is an order of magnitude below the temperature increase needed for thermal neural modulation16,17 or tissue overheating. Therefore, the film is unlikely to thermally modulate retinal neuronal activity.

Photoacoustic modulation of the ex vivo retina
To evaluate retinal responses following photoacoustic stimulation, we recorded the activity of retinal ganglion cells (RGCs) from ex vivo retinae of wild-type Long-Evans (LE) rats on a multi-electrode array (n = 4 rats). The PDMS/CS/PDMS film was placed on the ex vivo retina against the photoreceptor layer. It was photoactivated by a 1030-nm pulsed laser delivered through a 200-µm optical fiber (Fig. 2a), which was successively moved at different positions between stimulations.

We applied 4.2-ns laser pulses at a repetition rate of 1.9 kHz (every 520 µs) for a burst duration of db = 10 ms, with a pulse energy of 10 µJ (Fig. 2b, top; power density P = 0.27 W.mm−2), yielding an estimated peak-to-peak ultrasound pressure of 0.12 MPa.

Photoacoustic stimulation evoked robust RGC responses in healthy LE retinae (Fig. 2b, c, top two panels). Individual RGCs were considered responsive or modulated if their firing rate significantly increased (excited response) or decreased (inhibited response) relative to baseline (Fig. 2c, e). 100 LE RGCs (78%) exhibited an alteration in activity upon PA stimulation (Fig. 2e) out of the 129 spontaneously active RGCs on the electrodes contained in a 300-µm-radius area centered on the laser spot. Responsive RGC activity was mainly increased (92% of responding RGCs, Fig. 2d, left). RGCs with an increased activity had a mean response firing rate of 66±3.7 Hz (Fig. 2f), and had a mean response latency of 51±34.2 ms (Fig. 2g). The response latency was inversely correlated with the firing rate (Supplementary Fig. S8a).

To investigate the potential of such photoacoustic stimulation for restoring vision, we then stimulated ex vivo retinae from blind P23H rats (n = 4 rats). Similarly to LE retinae, though with a lower fraction, 89 out of 229 P23H RGCs (39%) exhibited robust responses to photoacoustic stimulation (Fig. 2c–e), predominantly with increased activity (93% of responding RGCs). Compared to LE RGCs, P23H RGC firing rate was significantly lower following stimulation (29±2.88 Hz, Fig. 2f) and response latency was significantly increased (89±65 ms, Fig. 2g). Only 36% of P23H RGCs had response latencies below 45 ms18, which was significantly fewer than LE cells (39%, Fig. 2g). These results on the P23H rat retina demonstrate the ex vivo efficacy of photoacoustic stimulation in modulating retinal ganglion cells activity in degenerated retinae.

To rule out the possibility that the healthy LE retina was responding to the pulsed infrared laser light19,20, we applied direct laser pulses to the LE rat retina. Using identical laser conditions to those applied on the film, only 3.6±0.9% of recorded RGCs showed a modified activity following direct film stimulation, compared to 77±20% of recorded RGCs following on-film photoacoustic stimulation (Supplementary Fig. S8b). These results were further confirmed in in vivo LE rats (see below). Furthermore, we found that the PA film absorbs more than 99% of the infrared laser energy (Supplementary Fig. S3). These results confirmed that the infrared laser light filtered out through the implant was not sufficient to directly activate the retinal circuit in the healthy LE retina.

Mechanistic exploration of retinal mechanosensitivity
It is currently unclear which layers in the retina contribute to the mechanosensitivity of the retina. We first observed an increase in response latency when photoreceptors have degenerated in P23H rat retinae, compared to LE retinae (Fig. 2g). To assess if photoreceptors initiate the short-latency US response, we bath-applied the group III mGluR agonist L-AP4 to LE retinae (Fig. 3a–c), which blocks the synaptic transmission between photoreceptors and ON bipolar cells21. We subsequently applied the kainate agonist ACET, which blocks most of the remaining glutamatergic synapses.

Before application of the blockers, a large fraction of recorded RGCs was responsive to the PA stimulation (58%, Fig. 3a, No blocker), with strong response firing rates (Fig. 3b, fr basal = 18±2 Hz, fr stim = 103±9 Hz, P<0.001, two-sided Wilcoxon). Following L-AP4 application, fewer RGCs were responsive to PA stimulation (19%, Fig. 3a, L-AP4), and their firing rates following stimulation were highly decreased (Fig. 3b, L-AP4: fr basal = 9±1 Hz, fr stim = 37±6 Hz, P<0.001, two-sided Wilcoxon). Moreover, L-AP4 predominantly suppressed short-latency responses (<45 ms, 7% of L-AP4-responsive cells, Fig. 3c, L-AP4). Following wash out of L-AP4, cell responses to PA stimulation were recovered (Fig. 3a, b, 40% of cells responding, fr basal = 7±1 Hz, fr stim = 86±13 Hz). Notably, the short-latency responses were recovered after the washout of the L-AP4 (Fig. 3c, baseline and washout). The L-AP4 condition mimics what was observed with recordings of P23H rat retinae with degenerated photoreceptors (P = 0.993, two-sided Mann-Whitney U test). These results further support that photoreceptors generate most of the short-latency RGC responses to ultrasound.

Addition of ACET to L-AP4 further suppressed RGC responses to PA stimulation, resulting in fewer cells responding (L-AP4+ACET: 8%, L-AP4: 19%, control with no blockers: 55%, Fig. 3a) and with lower firing rates (fr basal = 6±1 Hz, fr stim = 16±3 Hz, Fig. 3b). This additional effect of ACET indicated that cells in the inner retina, upstream of RGCs, are also contributing to the production of the US-mediated RGC activation. These pharmacological data indicate that part of the RGC responses to PA stimulation rely on upstream signaling pathways. Taken together, these results demonstrate the important contribution of photoreceptors in the short-latency response to ultrasound stimulation, as shown by previous studies6,18, and reliance of PA-mediated stimulation on upstream signaling.

Finally, to isolate the contribution of RGCs in PA-induced responses in the P23H model, we bath-applied glutamatergic blockers (rs)-CPP and CNQX to P23H retinae (Fig. 3d, e). PA-induced responses were nearly completely abolished, but not recovered following the washout of the blockers (basal: 24%, CPP+CNQX: 8%, washout: 8%, Fig. 3e). The lack of recovery during washout could be attributed to the difficulty of removing CPP and CNQX. These results suggest that the main mechanosensitive cells are upstream of RGCs, consistent with previous studies6,12,18,22, and that glutamate neurotransmission is required to transfer the mechanosensitive signal to the RGCs.

Dependence of RGC response on laser conditions
We further investigated RGC responses to photoacoustic stimulation using different laser repetition rates (10 µJ per pulse, f rep1 = 1.9 kHz with P 1 = 0.27 mW.mm−² and f rep2 = 3.5 kHz with P 2 = 0.52 mW.mm−²) and burst durations (db = 5-30 ms). In LE retinae, the firing rates of cells with excited responses increased with burst durations up to db = 25 ms for f rep1, while at f rep2 they plateaued up to db = 15 ms and decreased with longer burst durations (Fig. 4a, b, left). P23H RGCs with excited responses also showed an increase in firing rate with longer burst durations for f rep1 (Fig. 4a, b, right), while firing rate increased for burst durations up to db = 20 ms then decreased with longer burst durations for f rep2 (Fig. 4b, right). LE RGC firing rates were significantly higher than those for P23H RGCs, up to 2.8-fold during db = 25 ms and up to 4.7-fold during db = 20 ms with f rep1 and f rep2, respectively (Fig. 4b). These results suggest that the degenerated retina requires stronger photoacoustic stimulation, consistent with previous findings concluding on a higher acoustic stimulation threshold in degenerated retinae compared to wild-type retinae8. LE RGC response latencies did not increase with burst duration or repetition rate (Fig. 4c), nor did P23H RGC latencies.

Spatial resolution of ex vivo photoacoustic retinal stimulation
To investigate the spatial resolution of the photoacoustic retinal stimulation, we sequentially targeted multiple positions on the film by moving the laser-delivering 200-µm fiber at different sites above the photoacoustic film and mapped the responsive retinal cells (Fig. 5a). The laser repetition rate was set at f rep1 = 1.9 kHz for LE retinae (n = 11 sites) and f rep2 = 3.5 kHz for P23H retinae (n = 6 sites), to account for the higher modulation threshold previously described for P23H retinae.

To assess the spatial distribution of PA-modulated RGCs with excited responses, we mapped the maximum RGC firing rate relative to the stimulation site (Fig. 5b). For both LE and P23H RGCs, the maximum firing rates were located within an area slightly larger than the laser spot (<400 µm from center), and were negatively correlated with the distance from the laser spot (Fig. 5c). Furthermore, the percentage of responsive RGCs decreased when increasing the distance from the center of the laser spot (Fig. 5d). 73% of LE RGCs and 70% of P23H RGCs were modulated within a 100-µm distance, compared to 14% of LE RGCs and 6% of P23H RGCs at a 400-µm distance. These results indicate that stimulation with the PA film induces a localized response and demonstrate the potential for a high spatial resolution in photoacoustic stimulation.

In vivo safety of the photoacoustic implants
To test the feasibility of photoacoustic stimulation and the biosafety of the film in vivo, we chronically implanted 1-mm-diameter PA films in the subretinal space of LE and P23H rats. To investigate whether there is a risk of increased degeneration of inner retinal layers with the 115-µm-thick PDMS/CS/PDMS film, we also prepared and studied a thinner PDMS-CNT film. The PDMS/CS/PDMS film, used in the previous ex vivo experiments, was optimized for high photoacoustic conversion efficiency and easy handling. A thinner 40-µm-thick uniformly mixed PDMS-CNT film (characterized in Supplementary Fig. S4) was designed to approach the 30-µm thickness of the clinically tested PRIMA photovoltaic implant, which has shown no long-term adverse effects aside from minor retinal thinning in patients4. Both types of films were treated with oxygen plasma to make them hydrophilic, in order to improve cell adhesion and thus minimize the risks of implant displacement.

After implantation, eye fundus imaging confirmed the correct positioning of the implant near the optic nerve and the overall integrity of the retina (Fig. 6a, b). No complications, such as retinal tearing after implantation or major inflammation after 7 days post-implantation (dpi), were observed on the OCT images and eye fundus exams. Although the presence of glial cells between the implant and the retina is expected, stimulation efficacy should not be affected, as acoustic attenuation is below 10 dB.mm−1 23.

On OCT, the average retinal thickness was 174.0±2.3 µm for LE rats (n = 13) and 72.3±2.3 µm for degenerated P23H rats (n = 8). At the PDMS/CS/PDMS implant position, LE retinal thickness decreased to 123.2±2.9 µm at 15 dpi, 121.5±4.2 µm at 30 dpi, and 107.3±2.0 µm at 90 dpi (Fig. 6c). In LE rats implanted with the PDMS-CNT implant, similar values of 113.0±4.8 µm at 15 dpi and 105.8 µm at 30 dpi were measured (Fig. 6c). The decrease in the retinal thickness above the implant in LE rats was likely due to photoreceptor degeneration (Fig. 6a, right), caused by the physical separation of photoreceptors from the retinal pigment epithelium, as previously reported with other prostheses24,25. In implanted P23H rats, retinal thickness above the implant remained stable and comparable to the neighboring area for up to four months for both PDMS/CS/PDMS and PDMS-CNT implants (Fig. 6d).

Finally, we performed immunohistochemistry assays on LE (n = 3) and P23H (n = 4) rats. We confirmed the absence of rods and cones at the level of the implant in LE rats (Fig. 6e, i). We also observed the presence of activated microglia at the implantation site in both LE (Fig. 6e, ii) and P23H rats (Fig. 6f, ii), and of activated Müller glia only in LE rats (Fig. 6e, iii; P23H: Fig. 6f, iv). No inflammation was observed in the zone surrounding the implant (Fig. 6e, f, iv), except for a highly inflamed zone (Fig. 6e, iv, bottom right corner), which corresponded to the insertion track of the implant during surgery. Future studies will need to refine the implantation surgery of the PA implant to minimize inflammation caused by the insertion of the implant. Surface modification of the PDMS may also be explored to minimize inflammation at the level of the implant, in particular to reduce potential fibrosis.

Photoacoustic retinal stimulation in vivo
We then examined in vivo photoacoustic stimulation of the degenerated retina using subretinal PDMS/CS/PDMS and PDMS-CNT implants in LE rats. As mentioned in the previous section (Fig. 6a, c), the local detachment of the retina from the retinal pigment epithelium (RPE) caused by the implant induces local degeneration of photoreceptors in LE rats at the location of the implant, creating a localized model of retinal degeneration at the photoacoustic stimulation site (Fig. 6e, f). Activation of the visual pathway was assessed in the contralateral Superior Colliculus (cSC) using functional ultrasound imaging (fUSI), which measures relative changes in cerebral blood volume (rCBV) triggered by neuronal excitation (Fig. 7a). We chose to record the cSC instead of the primary visual cortex (V1) since anesthesia adversely affects the capability of fUSI to detect activation in V126,27. A cranial window was generated just prior to the experiment, preventing a long-term follow-up on the colliculus activity. To verify the coordinates of the cSC, we measured its natural visual activation using control full-field white light stimulation of the implanted eye (P = 0.02 mW.mm−2). This generated a larger CBV response area in the cSC (Fig. 7c, i), which was used as a reference area in further analyses (Fig. 7f). We measured a large increase in the amplitude of the rCBV, averaged on a 29-by-29-pixel (29 pixels = 302±10 µm) square region of interest (ROI; Fig. 7f, light gray), which was centered on the maximum responses in the cSC. The same ROI was subsequently used to compute the averaged rCBV for all of the stimulation conditions (Fig. 7f). To better define the expected size of the activated area for PA stimulation, we focused a 400-µm-diameter spot of 595-nm laser light (P = 0.21 mW.mm−2) onto the healthy retina next to the implant (Fig. 7b, iii). It similarly triggered an increase in rCBV in the cSC (Fig. 7c, ii), with an activated area representing 32±11% (n = 6) of the area activated by the full-field white light (Fig. 7f). In the same 29-by-29-pixel ROI, the rCBV amplitude and response kinetics were similar to those generated by the full-field white light stimulation (Fig. 7g, orange).

We then proceeded with photoacoustic stimulation on the implants (Fig. 7b, ii; Fig. 7d, top). The 1030-nm laser delivered eight 125-ms bursts during 2 s, repeated every 15 s, for a total of 15 stimulations per recording (Fig. 7d). Laser power densities were P = 0.29±0.06 W.mm−² (mean±SD) for PDMS/CS/PDMS implants and P = 0.39±0.12 W.mm−² for PDMS-CNT implants. The estimated peak-to-peak acoustic pressures at the surface of the implants were 0.05 MPa and 0.15 MPa for PDMS/CS/PDMS and PDMS-CNT implants, respectively. We observed activation in a large cSC area following each photoacoustic stimulation (Fig. 7d, e) using both types of implants (Fig. 7c, iii). Photoacoustic stimulation with the PDMS/CS/PDMS and PDMS-CNT implants activated cSC areas measuring 25±4% (n = 4 recordings) and 38±10% (n = 5 recordings) of the full-field-white-light-activated area, respectively (Fig. 7f). Furthermore, in the 29-by-29-pixel ROI, the averaged rCBV amplitudes for the PDMS/CS/PDMS (Fig. 7g, blue) and PDMS-CNT (Fig. 7g, black) implant stimulations reached similar amplitudes to that generated with the 400-µm-diameter spot of the 565-nm laser light on the healthy retina (Fig. 7g, orange). The size of the activated areas generated by stimulation with the PDMS/CS/PDMS and PDMS-CNT implants, and with the 595-nm-laser spot, were not significantly different (Fig. 7f). By contrast, a direct 1030-nm-light stimulation of the healthy retina (Fig. 7c, iv; P = 0.56±0.21 W.mm-2, 400-µm-diameter spot) did not generate a significant cSC activation (Fig. 7f). The averaged rCBV was negligible in the ROI, and statistically different from all of the other measurements (Fig. 7g, dark red). Taken together, these results show that the photoacoustic stimulation of the degenerated retina elicits a robust and local activation of the visual pathway downstream of the retina, with an amplitude and size comparable to a visible light stimulation of the retina.

Methods
Design of the photoacoustic film
Both the laser wavelength and the PA materials were optimized for safety and performance. Light wavelengths ranging from 500 nm to 1150 nm have maximum transmission in the human eye media43. A nanosecond laser with a 1030-nm wavelength was chosen to maximize transmission to the retina43, while avoiding triggering responses in photoreceptors, as AMD patients may retain peripheral vision. CS and CNT were selected as the absorber material due to their high photoacoustic conversion efficiency, accessibility, and lower safety concerns compared to lead-containing materials44,45. For the thermal expansion material, PDMS was identified as the best option due to its transparency, high Grüneisen parameter, excellent biocompatibility, and stability46. Although CS-PDMS films have been developed as laser ultrasound transducers28–30, we have engineered them into a sandwich-structure specifically designed as biomedical implant. The PDMS mixing ratio was adjusted to 5:1 to increase the Young’s modulus, thereby enhancing photoacoustic conversion efficiency47. The central frequency of our film is an intrinsic property of our film under our given experimental conditions. Several factors influence the ultrasound frequency generated by a photoacoustic device, including the laser pulse duration, the Grüneisen parameter of the elastomer material, and the effective optical absorption depth. In our setup, the laser pulse duration is fixed, PDMS is the only choice of absorber because it is both transparent and exhibits a high Grüneisen parameter, and the concentration of the CS layer remains constant due to the constraints of the flame synthesis process.

Fabrication of the photoacoustic films
To fabricate the 115-µm-thick PDMS/CS/PDMS film, a uniform layer of candle soot was flame-synthesized and deposited onto a glass slide for 20 s, achieving a thickness of approximately 4 µm. Subsequently, a degassed PDMS mixture of silicone elastomer base and curing agent (Sylgard 184, Dow Corning Corporation, USA) with a mix ratios of 5:1 was spin-coated at 500 rpm onto the candle soot layer, and cured at 110 °C for 15 min. The resulting cured film was then detached from the glass slide, inverted, and reattached. Another layer of PDMS mixture was spin-coated at 500 rpm and cured at 110 °C for 15 min.

To fabricate the 40-µm-thick PDMS-CNT film, we employed a recipe derived from previous work10. We initially prepared PDMS at mix ratios of 10:1. Subsequently, a 15% wt of CNT (<8 nm OD, 2–5 nm ID, length 0.5–2 µm, VWR, Inc., USA) was mixed with the PDMS, with the addition of IPA to facilitate CNT dissolution. The resulting mixture underwent a 5-minute sonication process, followed by a 30-minute degassing step to eliminate bubbles and IPA. The prepared mixture was then spin-coated onto a glass substrate at 500 rpm for 5 minutes. The coated substrate was cured at 110 °C for 15 min.

Both PDMS/CS/PDMS and PDMS-CNT films were treated with oxygen plasma for 1 min on both sides, to make the implant surface hydrophilic48. This allows for better cell adhesion, which reduces the risks of film displacement after the implantation surgery. The film was cut into smaller areas (5 × 5 cm²) and stored in distilled water before use to avoid reversion to their hydrophobic state. Biopsy punches (Kai medical) of 1 mm and 1.5 mm were used to cut individual photoacoustic films for ex vivo and in vivo experiments.

Characterization of the photoacoustic properties of the films
The photoacoustic properties of the films were characterized with a 40-µm needle hydrophone system (NH0040, Precision Acoustics Inc., UK) or an 85-µm needle hydrophone system (HGL-0085, Onda Corporation, USA). Illumination was provided by a Q-switched diode-pumped laser with a pulse width of 8 ns (RPMC, wavelength 1030 nm, repetition frequency 2.9 kHz, USA), delivered to one side of the film via a multimode fiber with a 200-µm core (FT200UMT, Thorlabs, USA). On the other side of the film, the hydrophone was mounted on a 3D stage and aligned with the area illuminated by the optical fiber. The signals were amplified with a pulser-receiver (Olympus, Model 5073PR, USA) and then recorded via a digital oscilloscope (Rigol, DS4024, USA).

Mapping the photoacoustic pressure field
Photoacoustic field microscopy was used to map the generated ultrasound field (Supplementary Fig. S7, Fig. 1a from Chen et al.16), as previously reported16. Here, a 1064-nm pulsed laser (OPOLETTE 355 LD, OPOTEK, pulse duration 5 ns) was used as the pump beam. A continuous wave 1310-nm laser (1310LD-4-0-0, AeroDIODE Corporation) served as the probe. A piece of PDMS/CS/PDMS film was mounted on a 50-µm optical fiber (FG050UGA, Thorlabs), and the 1064-nm laser was delivered to the film sample to generate the photoacoustic signals. A translation stage (ProScan III, Prior) was used to scan the generated ultrasound field. Under a single ns pulse, the PA-induced refractive index change was detected as the imaging contrast.

Temperature measurements
A J-type thermocouple with a 200-µm tip was set against the PDMS/CS/PDMS film inside 3% agarose gel, typically used for mimicking tissue49. The PDMS/CS/PDMS film was attached to a 200-µm optical fiber to assure the alignment between the illuminated area on the film and the thermocouple tip. A Q-switched diode-pumped laser with a pulse width of 4.5 ns (RPMC, wavelength 1030 nm, USA) was used to illuminate the film. The temperature on the film was recorded with a 2 kHz sampling rate from 10 recordings. Average data was computed from the 3 recordings that showed the highest temperature rise. Photos of the setup are shown in Supplementary Fig. S5. No transient temperature events faster than the 2 kHz acquisition frequency are expected to occur (Supplementary Fig. S6).

Animals
Wild-type Long-Evans male rats aged between 2 and 8 months were obtained from Janvier Laboratories. P23H male and female transgenic rats (9-14 months old) were raised locally. P23H rats serve as a model for autosomal dominant retinitis pigmentosa50.

Ex vivo experiments
Ex vivo retina preparation and blockers. The following procedures were carried out under dim red light. Animals were dark adapted for 30 min, then anesthetized with CO2 and euthanized by cervical dislocation. The eyes were enucleated and hemisected in carboxygenated (95% O2, 5% CO2, Air Liquide) Ringer medium containing (in mM): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaHPO4, 20 glucose, 26 NaHCO3, 1 CaCl2 and 0.5 L-Glutamine (Sigma-Aldrich) at pH 7.4. The medium was continuously perfused in the recording chamber at a speed of 1.5 mL.min-1 and was kept at around 37 °C.

Isolated retinae were placed on a dialysis membrane (Spectra/Por® 6 50 kD dialysis membrane, Spectrum) coated with poly-L-lysine (0.1%, Sigma-Aldrich), with the photoacoustic film between the dialysis membrane and the retina, and with photoreceptors against the film. The retinae were pressed against a multi-electrode array (MEA) (MEA256iR-ITO; Multi-Channel Systems, Reutlingen, Germany) with a custom 3D-printed piece.

Pharmacological blockers were bath-applied through the perfusion line. AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Tocris Bioscience) and NMDA glutamate receptor antagonist (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid ((RS)-CPP, 10 µM, Tocris Bioscience) were used to block signaling upstream of RGCs and therefore isolate the contribution of RGCs in PA-induced responses. Group III metabotropic glutamate receptor agonist L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4, 20 µM, Tocris Bioscience) was used to block the signaling between photoreceptors and ON bipolar cells21 to better understand the contribution of photoreceptors in retinal mechanosensitivity. Kainate antagonist (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxy-5-phenylthiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (ACET, 1 µM, Tocris Bioscience) was used to block remaining PA responses after L-AP4 application.

Ex vivo photoacoustic retinal stimulation
Photoacoustic stimulations were done with a 1030-nm, 4.2-ns-pulsed laser (One DPSS, Bright Solutions) delivered through a 200-µm-core 0.22 NA multimode SMA/SMA optic fiber (Thorlabs Inc, USA., ref M25L01). A second 200-µm-core was connected to the first fiber using a fixed attenuator (Thorlabs Inc., USA, ref FA26M) to control the power density. The optical fiber was inserted into a custom 3D-printed holder incorporated in a motorized XYZ stage with 5-nm precision (Sensapex, uMp-3 micromanipulator). It was lowered above the PA film at a ~90° angle and placed at a fixed (~ 1 mm) distance above the PA film. The illumination spot was measured ~300 µm in diameter on the film using ImageJ and the MEA electrode pitch as reference. A low-power 650-nm guiding beam (FIBERCHECK, Laser Components) was used to calibrate the beam position relative to the MEA.

Laser pulse repetition rate and the laser burst trains were controlled using a Teensy microcontroller custom written software (C++, Java, Python). In a typical stimulation, the laser delivered 10 µJ pulses with a repetition frequency f rep of 1.9 kHz or 3.5 kHz during a single 5-ms to 30-ms burst, repeated at 1 Hz for 40 bursts. PA film integrity was confirmed by the lack of photoelectric effect in the MEA recordings (Supplementary Fig. S7).

Analysis of MEA recordings
MEA raw traces were recorded through the MEA software (MCRack, Multichannel Systems). Spikes were sorted using SpyKING CIRCUS51, manually curated using phy52, and attributed to individual cells. Spikes were referenced relative to stimulus onset and grouped across trials in bins using a sliding window (bin width = 20 ms, increments = 5 ms). Cell activity in each bin was estimated using bootstrap resampling (n = 1000 resamples, 99% confidence intervals), and considered significantly increased or decreased if there were no overlapping confidence intervals compared to baseline (200 - 100 ms before stimulus onset).

RGCs were considered responsive or modulated if their firing rate was significantly increased or decreased compared to baseline for at least 15 ms consecutively, and response latency was defined as the first bin of this series. Noise clusters were filtered from the cell clusters by excluding cells with response latencies below 5 ms. A 300-µm-diameter area was illuminated by the 1030-nm laser (200-µm fiber) during photoacoustic stimulation. For quantifying responsive cells and dose responses (Figs. 2 and 3), we included only cells within 300 µm of the center of the illuminated area (“stimulation site”). The number of recorded cells per stimulation site is shown in Supplementary Fig. S8c. Cells with a response latency above 250 ms were excluded, as they were likely not a result from direct stimulation.

To analyze the relation between cell modulation and distance from the stimulation area (Fig. 5a), we calculated local averages in firing rate by assigning the firing rate of each cluster to a bin in a grid with a spacing of 25 µm, and smoothing the resulting averages using a convolution with a Gaussian kernel (sigma = 100 µm).

In vivo experiments
Successful implantation was defined as good positioning of the 1-mm-diameter film in the subretinal space, with no complications due to surgery. n = 8 adult (9-10 mo) P23H rats were successfully implanted and used for biocompatibility studies. n = 7 adult Long-Evans rats were successfully implanted at 8 weeks of age and used for photoacoustic stimulation.

Surgery procedures for chronic subretinal implantation
A 1-mm-diameter PA film was surgically placed in the subretinal space in the central region next to the optic nerve, as previously described27. Briefly, a small sclerotomy was performed on the dorsal sclera tangential to the cornea. A gel of sodium chondroitin sulfate-sodium hyaluronate (Viscoat Alcon) was injected into the sclerotomy to generate a retinal detachment. The implant was then inserted below the detached retina in the subretinal space, targeting a location adjacent to the optic disk.

Ocular imaging
Eye fundus imaging (MICRON® IV, Phoenix, USA) and optical coherence tomography (Bioptigen® OCT system, Leica microsystems, Germany) were performed at 7 and 15 days post-implantation (dpi) for all rats to monitor inflammation and confirm correct implantation. Additional imaging was conducted at 30, 60, 90 and 120 dpi for rats that did not undergo prior retinal stimulation.

Cranial window acute surgery
Anesthesia was provided with an intraperitoneal injection of 40 mg.kg-1 ketamine (Axience, France) and 0.14 mg.kg-1 medetomidine (Domitor®, Vétoquinol, France) diluted in sodium chloride. The animal was placed on a stereotaxic frame to perform a left craniotomy. Drops of ocular gel (Lubrithal®, Dechra, France) were applied and the eyes were then covered with a black cloth for dark adaptation. A rectangular piece of bone was removed from Bregma -3 mm to -8 mm.

Retinal stimulation and brain imaging
Retinal stimulation was performed 26–40 days after implantation surgery for rats with PDMS/CS/PDMS implants and 23–29 days after implantation surgery for PDMS-CNT implants, and immediately after the cranial window surgery. Anesthesia was re-administered every 45 min with one-third of the initial dose, up to a maximum of 5 injections. At the end of the experiment, the animals were euthanized using an intracardiac injection (Exagon®, Axience, France).

For full field light stimulations with a white LED source, the light power on the retina was estimated to be ~0.02 mW.mm−², based on the power entering the pupil of 1.2 mW. The choice of the stimulation protocol was informed by prior retina studies using functional ultrasound imaging27,53. Each 1.8-s stimulation sequence consisted of 6 evenly spaced 300-ms illuminations (LED on), repeated 15 times.

For 595 nm, 1030 nm light stimulation and photoacoustic stimulation, focused laser spots were aimed using a laser injector from the MICRON® 810-nm Image-Guided Laser modality combined with a MICRON® III camera (Phoenix, USA). A low power 650-nm guiding beam (FIBERCHECK, Laser Components) was coupled to the injector to safely choose the area to stimulate. The rat’s implanted eye was covered in ocular gel (Lubrithal®, Dechra, France) and in contact with the camera lens. Stimulation sequences for all 3 modalities were identical.

For 595-nm (continuous) laser stimulation, power density on retina was 26 µW in a 400±26 -µm-diameter laser spot. For photoacoustic stimulation, the same 1030-nm pulsed laser used in the ex vivo experiments was employed. The laser energy exiting the laser injector was Ep = 15 µJ per pulse. To aim at the implant for photoacoustic stimulation, the laser focal spot was not placed on the optical axis of the injector lens, which resulted in a loss of power. All the laser diameter at 1/e² (DL) and laser power density PL were estimated from average intensity profiles extracted with Fiji/ImageJ (Supplementary Figs. S9, S10) and are expressed as the mean ± standard deviation.

For 1030-nm laser stimulation on the retina: DL = 470±70 µm, PL = 0.56±0.21 W.mm-2. For 1030-nm photoacoustic stimulation with PDMS-CNT implants: DL = 360±60 µm, PL = 0.39±0.12 W.mm-2. For 1030-nm photoacoustic stimulation with PDMS/CS/PDMS implants: DL = 410±45 µm, PL = 0.29±0.06 W.mm−².

The pupil of the eye of interest was dilated with a tropicamide-based eye drop solution (Mydriaticum®, Théa, France) before the first recording. Body temperature was monitored with a rectal probe and maintained using a heating blanket. Respiratory and heart rates were continuously monitored (TCMT, Minerve, France). After local application of lidocaine (4 mg.kg-1, Laocaïne®, MSD, France), the thinned skull was exposed and covered with ultrasound gel. The rats were scanned with a system dedicated to small animal ultrasound neuroimaging (Iconeus, Paris, France). Doppler vascular images were obtained using the Ultrafast Compound Doppler Imaging technique54. The probe was positioned coronally at Bregma -6.5 mm in order to measure the cerebral blood volume (CBV) in the contralateral superior colliculus. Each frame was a compound plane wave frame55 resulting from the coherent summation of backscattered echoes obtained after successive tilted plane waves emissions. Then, the CBV signal was extracted from the tissue signal by filtering the image stacks with a dedicated spatiotemporal filter using Singular Value Decomposition56. Each transcranial Power Doppler image was obtained from 200 compounded frames acquired at 500 Hz frame rate.

Immunohistochemistry and image acquisition
After euthanasia, the eyes (n = 3 LE rats, n = 4 P23H rats) were enucleated and fixed with 4% paraformaldehyde (Cat no. 100496, Sigma-Aldrich) in 0.1 M phosphate buffer for 1 hour. The retinae were dissected and incubated in blocking solution (0.3% Triton X100 (Cat no. X100-500ML, Sigma-Aldrich), 10% Horse Serum (26050-070, Gibco) in phosphate buffer) with the primary antibodies (Iba1 (AB48004, Abcam), GFAP (Z0334, Dako), rhodopsin (MAB5316, Merck Millipore), cone arrestin (AB15282, Merck Millipore)) for three days at room temperature. The secondary antibodies used to detect immunolabeling were goat anti-IgG conjugated to Alexa-Fluor 488, 594 or 647 (A212026, A21203, and A21447, Invitrogen).

The retinae were flat-mounted, and imaged using an Olympus FV3000 confocal microscope with a 20X objective (UPLXAPO20X NA 0.8 WD 0.6, Evident).

Analysis of in vivo experiments
Analysis of OCT images. Mean retinal thickness next to and above the implant were measured with ImageJ on OCT images (diametral slices). The number of rats imaged at 30 dpi and later was lower than the number imaged at 7 dpi and 15 dpi because rats were used for terminal retinal stimulation recordings starting at 23 dpi.

Analysis of functional ultrasound imaging recordings
The correlation map of the CBV variations and the laser sequence for stimulation was computed by the manufacturer’s proprietary IcoStudio software (v1.5.2). A delay of either 2 or 3 s was computed in the calculation of the correlation to account for vascular delay (the chosen value maximizes the correlation). In correlation map displays (Fig. 7c), only significant pixels with a correlation threshold greater than 0.2 are shown. Maps with a correlation threshold of 0.1 are shown in Supplementary Fig. S11. A 29-by-29-pixel (29 pixels = 302±10 µm) region of interest (ROI) was defined for each animal, centered on the peak intensity of the correlation map for full-field-white-light stimulation. Relative CBV variations (rCBV) were extracted in this ROI for all stimulation types. For each recording (15 laser stimulations), the cerebral blood flow (CBV) was normalized into a relative steady-state value (rCBV) and calculated as the following: rCBV = (CBV(t) - CBV0) / CBV0, where CBV(t) is the power doppler value t seconds after the start of laser sequence, and CBV0 is the baseline in the ROI. The baseline was defined as the mean power doppler value 5 seconds before the start of the laser sequence. The data was bootstrapped to calculate confidence intervals.

Statistical analysis
Values are expressed as mean values ± standard error of the mean (SE) in figures and in the text, unless specified otherwise. Similarly, in scatter plots with error bars (Figs. 1, 4), data points and error bars represent the mean and the standard error of the mean, respectively. Statistical significance was analyzed with two-sided Wilcoxon signed-rank tests and two-sided Mann-Whitney U tests (Figs. 2–6). Pearson correlation (Figs. 2–4) was computed to quantify the strength and direction of the linear relationship between two continuous variables. One-way ANOVA was used to test the effect of a single factor on the mean of a dependent variable (Fig. 6). Finally, to estimate confidence intervals for a statistic of unknown distribution (average rCBV variations in Fig. 7) we used bootstrapped estimation (1000 samples, 95% confidence intervals). Statistical tests are provided in the figure legends.

Ethics
All animal experiments were conducted at the Paris Vision Institute, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals. Protocols were approved by the Local Animal Ethics Committee (Committee Charles Darwin CEEACD/N°5, project reference Apafis#40263-2023010909277429 v5) and conducted in agreement with Directive 2010/63/EU of the European Parliament.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper gives a comprehensive overview of the fabrication, characterization, and evaluation of the polydimethylsiloxane and carbon-based flexible film designed to convert near-infrared laser pulses into localized acoustic fields for mechanosensitive retinal stimulation. It aligns well with the current need for high-resolution, non-invasive retinal prosthetic solutions for vision restoration. However, there are some areas that require further clarification and refinement. Below are some critical comments, each pinpointing a specific issue in the text:\n1. For the introduction, in the 75 line, the term \"mechanosensitive retina\" is not commonly used in retinal physiology. Consider using a more precise phrase to describe mechanosensitivity in this context.\n2. The rationale for selecting 42.2 MHz as the stimulation frequency should be more thoroughly discussed. Prior studies suggest that high-frequency ultrasound may lead to overheating or suppression of neuronal activity. Could the authors provide further discussion for this frequency selection?\n3. The manuscript demonstrates high spatial resolution (≤50 µm) with fiber-based stimulation in proximity to the photoacoustic (PA) film and a resolution of <400 µm in ex vivo retina using a 200-µm fiber. However, in-vivo applications, where the laser must traverse the cornea and lens, refraction and scattering may significantly alter the final resolution. Could you consider simulating these optical and acoustic effects to predict the actual resolution in a whole-eye scenario for both rats and potential human eye applications (your current simulation result in sup materials is so simple).\n4. In the photoacoustic modulation of the ex vivo retina experiments, how do the authors ensure that the responses in LE rat retinae are not due to direct light activation of photoreceptors rather than photoacoustic effects? Further discussion or additional control experiments would be valuable. (For example, the authors could orient the PA film in the opposite direction, preventing ultrasound transmission to the retina while still blocking most of the laser light. This would allow assessment of whether any residual laser transmission alone can induce RGC responses.)\n5. The P23H rat model exhibits a reduced fraction of RGCs with short-latency responses compared to LE rats under ultrasound stimulation. Could the authors expand the discussion to provide potential mechanistic explanations for this difference?\n6. The study suggests that RGC responses originate from upstream retinal circuitry rather than direct activation. However, can the authors rule out direct PA-induced RGC activation? Additional discussion would be beneficial.\n7. The fUS results in the SC are compelling. Have the authors investigated whether similar activation occurs in the visual cortex or LGN?\n8. The manuscript describes LE rats as the wild-type (WT) control and P23H rats as the degeneration model, yet Lines 318-319 refer to \"photoreceptors degenerate LE rats.\" This appears inconsistent with earlier descriptions and should be clarified for coherence.\n9. The safety evaluation includes 90-120 day OCT monitoring, but OCT resolution is limited in detecting inflammatory responses or cellular damage. Could the authors consider adding immunofluorescence staining to assess inflammation and cellular responses in greater detail? Selecting a few inflammatory markers for whole-mount staining could provide a more comprehensive evaluation of biocompatibility.\n10. The manuscript does not detail whether specific methods (e.g., biocompatible adhesives or fixation strategies) were used to prevent PA film displacement post-implantation. Clarifying this would improve the clinical translatability of the approach.\n11. The study provides evidence of short-term biosafety (up to 120 days), but it does not address potential long-term immune responses or fibrosis formation that may compromise functionality over time. The authors mention that \"no major inflammation\" was observed (p. 17), but there is no quantitative assessment of immune markers such as glial activation or macrophage infiltration that could help substantiate this claim.\n12. The paper briefly mentions photovoltaic and ultrasound-based retinal prostheses (e.g., PRIMA), but the quantitative comparison of their system's spatial resolution, stimulation intensity, or field of view is not very clear relative to your photoacoustic film and other existing devices. Maybe you can add a comparative table in the supplementary.\n13. The author missed the statistical test description for latency differences for the distribution of response latencies (p. 9, Figure 2g).\n14. The author used functional ultrasound imaging (fUS) to measure cerebral blood volume (rCBV) in the superior colliculus as an indirect indicator of retinal stimulation. However, are there any baseline activity comparisons for the superior colliculus before and after implant? It makes it difficult to separate true stimulation effects from baseline vascular fluctuations.\n15. The author claim a lateral resolution of 56 µm, based on PA field mapping (p. 14, Fig. 4c). However, when reading this paper, it said the MEA electrode array used for RGC recordings has a 100-µm pitch, meaning that single-cell resolution cannot be accurately confirmed at the stated resolution. Please explain how this is confirmed because the true resolution might be much lower due to the averaging of responses across multiple electrodes.", "reviewer_2": "The work from Leong et al. proposes the technological translation to in vivo of a previous photoacoustic neuronal stimulation strategy for vision restoration.\nAlthough the device presents a very low energy conversion efficiency and it does not represent a novelty per se, the work is well structured and suitable for publication upon revision of a few important issues.\nFirst, the assessment of the toxicity/biocompatibility of the device should not be limited to the measurement of the retina thickness but include an immuno/histopathological study of the retina (i.e. Iba1 and GFAP, or CD86…, and photoreceptors markers).\nSecond, the in vivo results, although extremely solid, would be given more relevance by the employment of a dystrophic animal relying on a pathological degeneration of the photoreceptors with consequent rewiring of the inner retina. As mentioned before, although proven for other prosthetic solutions, IF/IHC of the retina below/above the implant should be included to prove the lack of light sensitivity.\nThird, it is not clear why there is a change in the architecture of the in vivo prosthesis for thickness purposes but then both candle soot and CNT prostheses are employed in the experiments for comparison. Please elaborate on the choice of device studied in vivo.\nMinor comments\n1. Figure 1b could be replaced by a SEM image better detailing the device. A top view could be also appreciated to immediately get the size of the prosthesis. Same thing for Supplementary Figure S4a.\n2. Figure 2i could benefit from a 2-way statistical analysis of variance.\n3. The title states “high precision” but indeed it is among photoacoustic ones, not overall. Might be misleading?", "reviewer_3": "In their manuscript, the authors describe a novel ultrasound-based retinal stimulation technology. They demonstrate that, by implanting a carbon-core film covered with polymethylsiloxane into the subretinal space in the rat eye, it is possible to generate photoacoustic waves inside the eye by stimulating the film with near-infrared light pulses, and that this mechanical stimulation can activate retinal neurons and downstream visual pathways in vivo. The topic of the study is very relevant, and the results achieved are of high interest, because they might open up a strategy for creating high-precision retinal prostheses with a wide field of view. The manuscript should be published after some revisions addressed below.\nThe manuscript is thoroughly thought and carefully written, particularly in the demonstration of photoacoustic modulation of the retina, both ex vivo and in vivo, as well as in healthy and degenerated retinas (without photoreceptor cells). However, the safety issues may be a bit less well addressed. My main concern is the photoacoustic waves potentially propagating towards the choroid from the film. As I understand it, the two sides of the films surrounding the carbon nanoparticle (and nanotube) layer were quite similar, suggesting that the photoacoustic waves generated by the rapid expansions of the film are likely to propagate in both directions, toward the retina on one side and toward the retinal pigment epithelium (RPE) and the choroid on the other side. Since the neuronal stimulation is presumed to occur through the activation of mechanosensitive channels in the retinal neurons, and RPE cells and the smooth muscles in vasculature contain several types of mechanosensitive channels as well, a discussion on the potential effects on the RPE and the choroid should be added in the Discussion section. Additionally, if necessary, what approaches could be used to dampen the photoacoustic waves towards RPE/choroid? The discussion should also cover the potential effects of photoacoustic stimulation on retinal blood vessels.\nAnother issue to address in Discussion is the question, how well mechanosensitivity is preserved in the retina. Could continuous “visual” mechanostimulation lead to some form of adaptation or depression of retinal mechanosensitivity? This information would be highly relevant for the potential development of photoacoustic retinal prostheses.\nSome other points to be considered:\nFig. 1g: The authors write that they measured the temperature “at the surface of the film”. With the robust size of the thermocouple (diameter 200 µm), especially when compared to the thickness of the retinas, the temperature values given may rather describe those in the near surroundings of the film.\nThe authors suggest on line 154 that photoreceptor cells may partly contribute to the ex vivo retinal mechanosensitivity. While it is not very essential for the study, blocking synaptic transmission from photoreceptor cells to bipolar cells using L-AP-4 would have provided a better comparison of the effectiveness of photoacoustic stimulation between the LE and P23H retinas by revealing the contribution of photoreceptors. Did the authors test the effect of L-AP-4 on mechanosensitivity in LE retinas?\nFig. 2 legend: (a) replace “on the outer layer” with “on the photoreceptor layer”\nLine 217: replace “requires higher thresholds for stimulation” with “requires stronger stimulation”\nLines 274-275: subretinal space might be mentioned\nLines 323-324: Were the pupils dilated during full-field white light stimulations?\nLine 387: The authors mention that “we developed flexible photoacoustic films...”. While true, similar PDMS-CS-PDMS films have been developed earlier, e.g. Chang et al.: Candle soot nanoparticles-polydimethylsiloxane composites for laser ultrasound transducers. Appl. Phys. Lett. 107, 161903 (2015). References to prior works should be added, also in Material and methods.\nLine 514: 5-nm instead of 0.5-nm"}, "author_response": "General statement\nWe would like to thank all of the reviewers for their valuable feedback and their insightful remarks.\nWe have tried to thoroughly address all of the concerns raised. In order to answer to important points\nthat were raised, we have performed additional experiments in which we\n1. performed immunohistochemistry in implanted LE and P23H rats, in order to better assess\nthe biocompatibility of our PA implant, and to evaluate the extent of photoreceptor\ndegeneration in LE rats induced by the subretinal implantation;\n2. applied L-AP4 and ACET pharmacological blockers to better assess the involvement of\nphotoreceptors in retinal mechanosensitivity.\nAll changes to the manuscript have been highlighted in red.\nFollowing a comment from Reviewer #2, we have decided to remove “high-precision” from our title,\nthus renaming our article “A flexible photoacoustic retinal prosthesis”.\nThe abstract was also slightly modified to fit the word limit.\nThe new experiments performed with pharmacological blockers led us to include a new subsection in\nthe Results section, entitled “Mechanistic exploration of retinal mechanosensitivity”. This section\nencompasses the new data with L-AP4 and ACET, as well as the data with (rs)-CPP and CNQX (previous\nFigure 2i). This data is presented in a new Figure 3, and all of the subsequent figures have been re-\nnumbered appropriately.\nMoreover, following the most recent meeting of fUSI-BIDS (functional UltraSound Imaging - Brain\nImaging Data Structure) users, the fUSI-BIDS community has chosen to adopt the acronym “fUSI”\ninstead of “fUS” to describe functional ultrasound imaging, in order to avoid possible confusion with\nthe “FUS” acronym, which stands for “Focused UltraSound”. We have therefore changed all acronyms\nof “fUS” into “fUSI”, in order to comply with those new guidelines (lines 322 and 359, and in Fig. 6a).\nResponse to reviewer comments\nReviewer #1 (Remarks to the Author):\nThe paper gives a comprehensive overview of the fabrication, characterization, and evaluation of the\npolydimethylsiloxane and carbon-based flexible film designed to convert near-infrared laser pulses\ninto localized acoustic fields for mechanosensitive retinal stimulation. It aligns well with the current\nneed for high-resolution, non-invasive retinal prosthetic solutions for vision restoration. However, there\nare some areas that require further clarification and refinement. Below are some critical\ncomments, each pinpointing a specific issue in the text:\n1. For the introduction, in the 75 line, the term \"mechanosensitive retina\" is not commonly used in\nretinal physiology. Consider using a more precise phrase to describe mechanosensitivity in this\ncontext.\nWe have replaced “mechanosensitive retina” with “mechanosensitive retinal cells” in line 75, and\nmodified the sentence to:\n“The generated ultrasound waves stimulates the mechanosensitive retinal cells6,8,12, resulting in a\nchange in retinal ganglion cell activity, which is then transferred to the brain.” (Lines 74-76)\n2. The rationale for selecting 42.2 MHz as the stimulation frequency should be more thoroughly\ndiscussed. Prior studies suggest that high-frequency ultrasound may lead to overheating or\nsuppression of neuronal activity. Could the authors provide further discussion for this frequency\nselection?\nThe ultrasound frequency of a photoacoustic device is determined by the effective absorption\ndepth of light. Previous work has demonstrated frequency control in the range of 1.0–5.5 MHz by\nadjusting the concentration of the absorber CNT1. In our case, the CS-based device is fabricated using\na flame synthesis process, which prevents concentration tuning for modifying the ultrasound\nfrequency. Consequently, the 42.2-MHz central frequency was not selected, but is an intrinsic\nproperty of this film.\nWe believe 42.2 MHz is suitable for retinal stimulation. Previous work by other groups\ndemonstrated ultrasound stimulation of the retina at 43 MHz required a lower intensity than a lower\nfrequency (15 MHz)2. Moreover, high ultrasound frequencies have a higher spatial resolution than\nlower frequencies, which allows us to preserve the spatial resolution of the optic fiber delivering the\nlaser light.\nRegarding the heating, we emphasize that photoacoustic neuromodulation involves a\ndifferent heat accumulation process than conventional ultrasound neuromodulation. In conventional\nultrasound applications, overheating concerns arise when operating at 42 MHz due to the continuous\nnature of ultrasound exposure. In contrast, photoacoustic neuromodulation generates only a single\nultrasound pulse (~40 ns, as shown in Figure 1C) every 0.16 ms at a 6 kHz repetition rate, resulting in\na duty cycle of just 0.025%. This indicates that heat accumulation from ultrasound exposure is minimal,\neven at 42 MHz. Instead, the primary source of heat in the photoacoustic process is laser absorption,\nwhich is more dependent on laser dosage than ultrasound frequency. In our case, as indicated in line\n105, we only observe an average heating of 0.52 ± 0.09 °C with our stimulation parameters, and\ntherefore conclude that the reported neuromodulation is likely not due to thermal activation.\nWe have added a more thorough discussion on the use of the 42.2-MHz frequency in the\nResults section and in the Material and methods:\n“This central frequency is an intrinsic property of our film under our experimental conditions, given\nthe fixed laser pulse duration, the properties of PDMS, and the constant concentration of the CS layer\nset by the constraints of the flame synthesis process.” (Results, lines 84-87)\n“The central frequency of our film is an intrinsic property of our film under our given experimental\nconditions. Several factors influence the ultrasound frequency generated by a photoacoustic device,\nincluding the laser pulse duration, the Grüneisen parameter of the elastomer material, and the\neffective optical absorption depth. In our setup, the laser pulse duration is fixed, PDMS is the only\nchoice of absorber because it is both transparent and exhibits a high Grüneisen parameter, and the\nconcentration of the CS layer remains constant due to the constraints of the flame synthesis process.”\n(Material and Methods, lines 543-549)\nWe also added an additional comment on tissue heating:\n“Ultrasound absorption could cause an additional increase in temperature, but it is expected to be\nmuch lower due to the very low acoustic energy.” (Lines 104-105)\n“This value is an order of magnitude below the temperature increase needed for thermal neural\nmodulation16,17 or tissue overheating.” (Line 111)\n1. Shi, L. et al. Non-genetic photoacoustic stimulation of single neurons by a tapered fiber\noptoacoustic emitter. Light Sci. Appl. 10, 143 (2021).\n2. Menz, M. D. et al. Radiation Force as a Physical Mechanism for Ultrasonic Neurostimulation\nof the Ex Vivo Retina. J. Neurosci. 39, 6251–6264 (2019).\n3. The manuscript demonstrates high spatial resolution (≤50 µm) with fiber-based stimulation in\nproximity to the photoacoustic (PA) film and a resolution of <400 µm in ex vivo retina using a 200-µm\nfiber. However, in-vivo applications, where the laser must traverse the cornea and lens, refraction and\nscattering may significantly alter the final resolution. Could you consider simulating these optical and\nacoustic effects to predict the actual resolution in a whole-eye scenario for both rats and potential\nhuman eye applications (your current simulation result in sup materials is so simple).\nThe current study does address the theoretical resolution of light projections in in-human\nconditions. While it is necessary to have models as proposed by the reviewer when designing an\nimplant for in-human applications, we want to emphasize that the goal of the current study is to\ndemonstrate a proof of concept of the feasibility of photoacoustic activation of the retina ex vivo and\nin vivo. Future studies are needed to measure the optimal resolution that could be achieved with this\nsystem.\nThe resolution of the laser injector we have used in vivo is 50 µm. While we expect some\nscattering and diffraction due to the imperfect light source and the cornea and lens, this resolution is\nenough to target the 1-mm-diameter implant and a zone outside of it, allowing us to show that the\ncSC activation we observe is indeed caused by PA stimulation of the retina, and not by the laser.\nWe therefore think that an advanced simulation model is out of the scope of the current study,\nespecially given the complexity of accurately modeling the eye for both optical and ultrasonic\napplications. The simulation in the extended data is designed to estimate the applied optical power at\nthe implant level, for which it suffices.\nWe added this potential limitation to the discussion, in lines 514-515:\n“Whole-eye models will be necessary to predict the actual resolution that could be achieved for in-\nhuman applications.”\n4. In the photoacoustic modulation of the ex vivo retina experiments, how do the authors ensure that\nthe responses in LE rat retinae are not due to direct light activation of photoreceptors rather than\nphotoacoustic effects? Further discussion or additional control experiments would be valuable. (For\nexample, the authors could orient the PA film in the opposite direction, preventing ultrasound\ntransmission to the retina while still blocking most of the laser light. This would allow assessment of\nwhether any residual laser transmission alone can induce RGC responses.)\nUnfortunately, we cannot perform the proposed experiment because the PA film emits\nacoustic waves on both sides of the implant, therefore inverting the implant would still apply US waves\nto the retina. However, we did address the question of a direct activation of the LE retina by showing\nthat even the same infrared laser intensity as the one applied to the film activated only 3.6 ± 0.9% of\nrecorded RGCs. This infrared-elicited activity, when applied directly off-implant, does not compare to\nthe 77 ± 20% following on-film photoacoustic stimulation (Fig. 2j, lines 167-170). Similarly, the off-\nimplant stimulation in in vivo LE rats did not generate a significant activation of the superior colliculus\n(Fig. 7f). Furthermore, we found that the PA film absorbs more than 99% of the on-film applied laser\nenergy (Supplementary Fig. S3, lines 170-171).\nThe text in lines 169-177 was modified to clarify this important result:\n“To rule out the possibility that the healthy LE retina was responding to the pulsed infrared laser\nlight18,19, we performed off-film stimulation by applying applied direct laser pulses directly to the LE\nrat retina. Using identical laser conditions to those applied on the film, only 3.6 ± 0.9% of recorded\nRGCs showed a modified activity following direct off-film stimulation, compared to 77 ± 20% of\nrecorded RGCs following on-film photoacoustic stimulation (Fig. 2ji). These results were further\nconfirmed in in vivo LE rats (see below). Furthermore, we found that the PA film absorbs more than\n99% of the on-film applied infrared laser energy (Supplementary Fig. S3). These results confirmed that\nthe infrared laser light filtered out through the implant was not sufficient to directly activate the retinal\ncircuit in observed RGC responses upon laser activation of the photoacoustic film were not caused by\na direct light activation of the healthy LE retina.”\n5. The P23H rat model exhibits a reduced fraction of RGCs with short-latency responses compared to\nLE rats under ultrasound stimulation. Could the authors expand the discussion to provide potential\nmechanistic explanations for this difference?\nFollowing the suggestion of reviewer #3, we have performed additional experiments to test\nthe contribution of photoreceptors to retinal mechanosensitivity by bath-applying L-AP4 and ACET to\nex vivo LE retinae (n = 2 retinae, 181 recorded cells, 101 PA-responsive cells before blockers). L-AP4 is\na group III metabotropic glutamate receptor agonist which blocks the signaling between\nphotoreceptors and ON bipolar cells3. ACET is a kainate antagonist which was used to block remaining\nPA responses after L-AP4 application.\nApplication of L-AP4 to LE retinae induced a reduction in the fraction of PA-responsive cells\n(19% vs 58% before L-AP4, Fig. 3b, L-AP4 and No blocker) as well as a decrease in response firing rate\n(from 103 ± 2 Hz to 37 ± 6 Hz, Fig. 3b, No blocker and L-AP4). Interestingly, L-AP4 blocked the fast\nresponses (<45 ms, Fig. 3c, L-AP4), with 55% of responsive cells having a fast response before blockers\nto only 7% after L-AP4. The shift in firing rate post-stimulation and in response latency generates a\nsituation similar to what we observed with P23H RGCs (p = 0.993, 2-sided Mann Whitney-U). Taken\ntogether, these results demonstrate the important contribution of photoreceptors in the short-\nlatency response to ultrasound stimulation. The difference in fraction of responding cells between\nP23H and LE can thus be explained by the partial dependency of RGC responses on photoreceptors in\nLE, which are absent in P23H retinae.\nFor clarity’s sake, we have modified the structure of our manuscript to add a section on\nsynaptic transmission blocking. Therefore, the previous Figure 2i has now been moved to Figure 3e.\nAn additional experiment has been done with (rs)-CPP and CNQX with a P23H retina in order to get a\nbetter significance of the results. Current Figure 3d-e takes into account this new experiment. The\nresults of the L-AP4 experiments are shown in Figure 3a-c. The new Figure 3 (page 13-14) is also shown\nbelow. The Material and Methods were also modified to include the use of L-AP4 and ACET, in lines\n627-631.\nThe following text was added to the results section, in lines 215-237:\n“Mechanistic exploration of retinal mechanosensitivity\nIt is currently unclear which layers in the retina contribute to the mechanosensitivity of the retina. We\nfirst observed an increase in response latency when photoreceptors have degenerated in P23H rat\nretinae, compared to LE retinae (Fig. 2g). To assess if photoreceptors initiate the short-latency US\nresponse, we bath-applied the group III mGluR agonist L-AP4 to LE retinae (Fig. 3a-c), which blocks the\nsynaptic transmission between photoreceptors and ON bipolar cells43. We subsequently applied the\nkainate agonist ACET, which blocks most of the remaining glutamatergic synapses.\nBefore application of the blockers, a large fraction of recorded RGCs was responsive to the PA\nstimulation (58%, Fig. 3a, No blocker) with strong response firing rates (103 ± 2 Hz, Fig. 3a, No blocker).\nFollowing L-AP4 application, fewer RGCs were responsive to PA stimulation (19%, Fig. 3b, L-AP4), and\ntheir firing rates following stimulation were highly decreased (37 ± 6 Hz, Fig. 3b, L-AP4). Moreover, L-\nAP4 predominantly suppressed short-latency responses (<45 ms, 7% of recorded cells, Fig. 3c, L-AP4).\nThese short-latency responses were recovered after the washout of the L-AP4 (Fig. 3c, baseline and\nwashout): The L-AP4 condition mimics what was observed with recordings of P23H rat retinae with\ndegenerated photoreceptors (p = 0.993, 2-sided Mann Whitney-U). These results further support that\nphotoreceptors generate most of the short-latency RGC responses to ultrasound.\nAddition of ACET to L-AP4 further suppressed RGC responses to PA stimulation (L-AP4 + ACET: 8 %, L-\nAP4: 19%, control with no blockers; 55%, Fig. 3b). This additional effect of ACET indicated that cells in\nthe inner retina, upstream of RGCs, are also contributing to the production of the US-mediated RGC\nactivation. These pharmacological data indicate that part of the RGC responses to PA stimulation rely\non upstream signaling pathways. Taken together, these results demonstrate the important\ncontribution of photoreceptors in the short-latency response to ultrasound stimulation, as shown by\nprevious studies6,18, and reliance of PA-mediated stimulation on upstream signaling.\nFigure 3. Photoacoustic responses under pharmacological blocking. (a) Percentage of cells\nmodulated by photoacoustic stimulation per stimulation site before and after L-AP4 application. No\nblocker: 49%; L-AP4: 16%; L-AP4 + ACET: 7%; washout: 40% (2 retinae, n = 9 stimulation sites). (b)\nGroup III mGluR agonist L-AP4 decreased RGC spontaneous firing rate and firing rate during PA-elicited\nresponses in LE retinae. The population firing rate of RGCs (n = 41 cells, 2 retinae) is compared between\nbaseline (Basal) and stimulation (Stim.), before blocker application (No blocker), following blocker\napplication (L-AP4) and after washout (Washout) with Ringer medium. Firing rates per condition, no\nblockers: fr = 18 ± 2Hz, fr = 103 ± 9 Hz (p < 0.001); L-AP4: fr = 9 ± 1 Hz, fr = 37 ± 6 Hz (p <\nbasal stim basal stim\n0.001); L-AP4+ACET: fr = 6 ± 1 Hz, fr = 16 ± 3 Hz (p = 0.024), washout: fr = 7 ± 1 Hz, fr = 86\nbasal stim basal stim\n± 13 Hz (p < 0.001). Comparison of the population stimulation firing rate fr after blocker application\nstim\nand fr before application (p < 0.001), and fr after washout (p < 0.001). (c) L-AP4 increases\nstim stim\nresponse latency in LE retinae. Response latency per condition, no blockers: 49 ± 5 ms; L-AP4: 65 ± 4\nms; washout: 42 ± 3 ms. Comparison of the response latency before blocker application and after (p <\n0.001, Wilcoxon signed-rank), and after washout (Friedmanchi square, p = 0.4). Due to absence of\nresponses during application of L-AP4+ACET blockers, no latencies are reported for this group. Dashed\nblack line: 45-ms cutoff for slow- and fast-latency responses. (d) Percentage of cells modulated by\nphotoacoustic stimulation per stimulation site before and after (rs)-CPP and CNQX application. No\nblocker: 42 %; (rs)-CPP + CNQX: 5 %; washout: 8 % (2 retinae, n = 5 stimulation sites). (e) Glutamate\nblockers (rs)-CPP and CNQX abolished RGC responses to PA stimulation in P23H retinae. The\npopulation firing rate of RGCs (n = 25 cells, 2 retinae) is compared between baseline (basal) and\nstimulation (stim), before blocker application (no blocker), following blocker application ((rs)-\nCPP+CNQX) and after washout (washout) with Ringer medium. Firing rates per condition, no blockers:\nfr = 11 ± 2 Hz, fr = 100 ± 23 Hz (p < 0.001); (rs)-CPP+CNQX: fr = 7 ± 2 Hz, fr = 23 ± 10 Hz (p\nbasal stim basal Stim\n=0.141); washout: fr = 8 ± 2 Hz, fr = 24 ± 9 Hz (p = 0.016). Comparison of the population\nbasal stim\nstimulation firing rate fr after blocker application and fr before application (p < 0.001, Wilcoxon\nstim stim\nsigned-rank), and fr after washout (p < 0.001, Wilcoxon signed-rank). For each panel, groups are\nstim\ntested for significant differences using Friedman chi square, with post-hoc Wilcoxon signed-rank. p-\nvalues are holm-corrected (***: p<0.001, **: p<0.05).”\n3. Thoreson, W. B. & Ulphani, J. S. Pharmacology of selective and non-selective metabotropic\nglutamate receptor agonists at L-AP4 receptors in retinal ON bipolar cells. Brain Res. 676, 93–102\n(1995).\n6. The study suggests that RGC responses originate from upstream retinal circuitry rather than direct\nactivation. However, can the authors rule out direct PA-induced RGC activation? Additional discussion\nwould be beneficial.\nWe bath-applied (rs)-CPP and CNQX to P23H retinae, which block all excitatory synaptic\nconnections to RGCs4 (Material and Methods, lines 622-626). Under this condition, any response\ninduced by PA-stimulation is due to direct PA-induced RGC activation because their excitatory inputs\nare suppressed. We observed an abolition of PA-induced responses when applying (rs)-CPP and CNQX,\nwith no significant difference of firing rate between the baseline and PA-stimulation conditions (Fig.\n3e). Additionally, our added data on the L-AP4+ACET blockers (Fig. 3a-c), which block photoreceptor\nsignaling, demonstrates the same effect. Very few RGCs remain responsive under the (rs)-CPP and\nCNQX or the L-AP4+ACET conditions, which strongly suggests that RGCs are not directly activated by\nphotoacoustic stimulation.\nWe have added a subsection in the results section (lines 202-229) on the mechanistic\nexploration of retinal mechanosensitivity (see response to question 5). The text was slightly modified\nto clarify the aim of using (rs)-CPP and CNQX (lines 238-239):\n“Finally, to isolate the contribution of RGCs in PA-induced responses in the P23H model, we bath-\napplied glutamatergic blockers (rs)-CPP and CNQX to P23H retinae (Fig. 3d,e).”\nThe Material and Methods section (lines 622-631) was also modified to clarify the effect of\n(rs)-CPP and CNQX, and to include the use of L-AP4 and ACET:\n“Pharmacological blockers were bath-applied through the perfusion line. AMPA/kainate glutamate\nreceptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μM, Tocris Bioscience) and\nNMDA glutamate receptor antagonist (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid\n((RS)-CPP, 10 µM, Tocris Bioscience) were used bath-applied through the perfusion line to block\nsignaling upstream of RGCs and therefore isolate the contribution of RGCs in PA-induced responses.\nGroup III metabotropic glutamate receptor agonist L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4, 20\nµM, Tocris Bioscience) was used to block the signaling between photoreceptors and ON bipolar cells43\nto better understand the contribution of photoreceptors in retinal mechanosensitivity. Kainate\nantagonist (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxy-5-phenylthiophene-3-yl-methyl)-5-\nmethylpyrimidine-2,4-dione was used to block remaining PA responses after L-AP4 application.”\n4. Khabou, H. et al. Optogenetic targeting of AII amacrine cells restores retinal computations\nperformed by the inner retina. Mol. Ther. Methods Clin. Dev. 31, (2023).\n7. The fUS results in the SC are compelling. Have the authors investigated whether similar activation\noccurs in the visual cortex or LGN?\nWe thank the reviewer for their positive comment on our fUSI results in the SC. We analyzed\nthe activity in the visual cortex during stimulation (Figure 3C, visual areas not marked+). We did not\nfind responses in either area, which we attribute to the alteration of blood flow during anesthesia,\nwhich reduces the fUSI signal in these specific areas. Indeed, a previous study has shown that\nanesthesia significantly reduces both the amplitude and the speed of hemodynamic responses in the\nvisual cortex5. This attenuation adversely affects the capacity of fUSI to detect activation in the primary\nvisual cortex (V1) following visual stimulation6. Furthermore, we opted to focus our recordings on the\nSC rather than the LGN because 85 to 90% of RGCs project to the SC in rodents7,8. Thus, we strategically\npositioned our imaging plane on the area where we recorded the maximal SC activation following\nwhite-light stimulation, rather than attempting to capture both the SC and LGN simultaneously. To\nanswer the reviewer in the manuscript, we specified our choice on the superior colliculus due to the\ndifficulty of recording fUSI signal in the visual cortex of anesthetized animals with the corresponding\nreferences, in lines 404-406:\n“ We chose to record the cSC instead of the primary visual cortex (V1) since anesthesia adversely\naffects the capability of fUSI to detect activation in V130,31.”\n5. Pisauro, M. A., Dhruv, N. T., Carandini, M. & Benucci, A. Fast Hemodynamic Responses in the\nVisual Cortex of the Awake Mouse. J. Neurosci. 33, 18343–18351 (2013).\n6. Provansal, M. et al. Functional ultrasound imaging of the spreading activity following\noptogenetic stimulation of the rat visual cortex. Sci. Rep. 11, 12603 (2021).\n7. Ellis, E. M., Gauvain, G., Sivyer, B. & Murphy, G. J. Shared and distinct retinal input to the\nmouse superior colliculus and dorsal lateral geniculate nucleus. J. Neurophysiol. 116, 602–610\n(2016).\n8. Seabrook, T. A., Burbridge, T. J., Crair, M. C. & Huberman, A. D. Architecture, Function, and\nAssembly of the Mouse Visual System. Annu. Rev. Neurosci. 40, 499–538 (2017).\n8. The manuscript describes LE rats as the wild-type (WT) control and P23H rats as the degeneration\nmodel, yet Lines 318-319 refer to \"photoreceptors degenerate LE rats.\" This appears inconsistent with\nearlier descriptions and should be clarified for coherence.\nThe photoreceptor degeneration mentioned in LE rats is due to the local detachment of the\nretina from the RPE at the level of the implant (line 345-347). This can be seen in the thinning of the\nretina at 15+ dpi (Fig. 6c) and in the newly added IHC staining in Fig. 6e (see the response to question\n9).\nTo reemphasize this degeneration in the following section, we have changed our sentence to:\n“As mentioned in the previous section (Fig. 6a, c), the local detachment of the retina from the retinal\npigment epithelium (RPE) caused by the implant induces local degeneration of photoreceptors\ndegenerate above the implant in LE rats at the location of the implant, creating a localized model of\nretinal degeneration at the photoacoustic stimulation site (Fig. 6e, f) (Fig. 5a, c).” (lines 398-402).\n9. The safety evaluation includes 90-120 day OCT monitoring, but OCT resolution is limited in detecting\ninflammatory responses or cellular damage. Could the authors consider adding immunofluorescence\nstaining to assess inflammation and cellular responses in greater detail? Selecting a few inflammatory\nmarkers for whole-mount staining could provide a more comprehensive evaluation of biocompatibility.\nWe thank the reviewer for their suggestion of adding immunofluorescence staining results.\nWe have done whole-mount immuno-histochemistry assays on 3 LE rats and 4 P23H rats. We stained\nfor microglia (Iba1), astrocytes (GFAP), rods (rhodopsin) and cones (cone arrestin).\nWe have observed the presence of activated microglia at the implantation site in both LE (Fig.\n6e, ii) and P23H rats (Fig. 6f, ii). Activated Müller glia were only present in LE rats (Fig 6e, iii; P23H: Fig\n6f, iv). We observed no significant inflammation (i.e. increase in GFAP expression and in microglia\nnumber or size) surrounding the implant (Fig 6e-f, iv), except for a zone which corresponds to its\ninsertion track (Fig. 6e, iv, bottom right corner).\nMoreover, we confirmed the degeneration of photoreceptors at the location of the implant\nin LE rats (Fig. 6e, i) with the loss of both the rhodopsin and the cone-arrestin immunolabelings. This\nobservation is consistent with the retinal thinning at the implant position after 15 dpi (Fig. 6c).\nWe have added the panels presented below to Figure 6 (Fig. 6e-f), added a part on\nimmunohistochemistry in the Material and Methods section, and added Clémence Bradic in the list of\nauthors, for her contribution to the histological results.\nFigure 6. In vivo photoacoustic implant biocompatibility. (e) Immunolabeling of an implanted LE\nretina, labeling rods (Rho, red, i, iv), cones (CAR, green, i, iii), microglia (Iba1, magenta, i, ii, iv), and\nMüller cells (GFAP, green, i, iii). i - iii: implanted zone. iv: zone surrounding the implant. (f) Same as (e)\nfor P23H, with microglia (Iba1, magenta, i, ii, iv) and Müller cells (GFAP, green, i, iii). Scale bar for (e)\nand (f) is 200 µm.\n10. The manuscript does not detail whether specific methods (e.g., biocompatible adhesives or\nfixation strategies) were used to prevent PA film displacement post-implantation. Clarifying this\nwould improve the clinical translatability of the approach.\nTo minimize displacement, both sides of the film were treated with oxygen plasma to make\nthem hydrophilic9. The films were then stored in distilled water until implantation the time of\nimplantation to maintain their hydrophilic properties, as described in the Material and Methods (Lines\n537-539). Indeed, hydrophilic surface modification of PDMS has been shown to improve cell adhesion\nfor cell culture10. We believed it to be sufficient to avoid implant displacement over three months of\nimplantation. Indeed, we did not observe any implant displacement after the implantation surgery.\nFor longer implantation durations, longer exposure to oxygen plasma or other chemical\ncoating of the PDMS11 may be warranted to better maintain the hydrophilic properties; however, such\nconsiderations extend beyond the scope of our current study.\nWe have added a sentence in the results section (Lines 347-349) and in the Material and\nMethods section (Lines 568-570) to address this concern.\n“Both types of films were treated with oxygen plasma to make them hydrophilic, in order to improve\ncell adhesion and thus minimize the risks of implant displacement.” (Resuts, lines 347-349)\n“Both PDMS/CS/PDMS and PDMS-CNT films were treated with oxygen plasma for 1 min on both sides,\nto make the implant surface hydrophilic39. This allows for better cell adhesion, which reduces the risks\nof film displacement after the implantation surgery.” (Material and Methods, lines 568-570)\n9. Duangkanya, K., Kopwitthaya, A., Chanhorm, S. & Infahsaeng, Y. Oxygen plasma\ntreatment time induced hydrophilicity of polydimethylsiloxane (PDMS) thin films for liquid lenses\napplication. Mater. Today Proc. 65, 2442–2445 (2022).\n10. Chuah, Y. J. et al. Surface modifications to polydimethylsiloxane substrate for stabilizing\nprolonged bone marrow stromal cell culture. Colloids Surf. B Biointerfaces 191, 110995 (2020).\n11. Jastrzebska, E. et al. Biological characterization of the modified poly(dimethylsiloxane)\nsurfaces based on cell attachment and toxicity assays. Biomicrofluidics 12, 044105 (2018).\n11. The study provides evidence of short-term biosafety (up to 120 days), but it does not address\npotential long-term immune responses or fibrosis formation that may compromise functionality over\ntime. The authors mention that \"no major inflammation\" was observed (p. 17), but there is no\nquantitative assessment of immune markers such as glial activation or macrophage infiltration that\ncould help substantiate this claim.\nAs mentioned in our response to question 9, we have performed new immunohistochemistry\nassays to evaluate inflammation after implantation. We have observed the presence of activated\nmicroglia at the implantation site in both LE (Fig. 6e, ii) and P23H rats (Fig. 6f, ii), and of activated\nMüller glia only in LE rats (Fig 6e, iii; P23H: Fig 6f, iv). We observed no significant inflammation\nsurrounding the implant (Fig 6e-f, iv), except for a zone which corresponds to the insertion track of\nthe implant during surgery (Fig. 6e, iv, bottom right corner).\nWe have added a sentence about potential solutions for long-term immune responses or\nfibrosis in Lines 374-375:\n“Surface modification of the PDMS may also be explored to minimize inflammation at the level\nof the implant, in particular to reduce potential fibrosis.”\nWe want to emphasize that this study is a first proof of concept study for PA stimulation for\nvisual restoration, and that future studies will focus more on material optimization for\nbiocompatibility purposes, as well as longer-term biosafety.\n12. The paper briefly mentions photovoltaic and ultrasound-based retinal prostheses (e.g., PRIMA),\nbut the quantitative comparison of their system's spatial resolution, stimulation intensity, or field of\nview is not very clear relative to your photoacoustic film and other existing devices. Maybe you can\nadd a comparative table in the supplementary.\nWe thank the reviewer for their suggestion of adding a comparative table. We’ve added the\nSupplementary Table T2, also shown below, to compare our work with current technologies.\nOur Argus II14 PRIMA POLYRETINA Ultrasound GenSight’s\nphotoacousti by Pixium 16 array17 GS03018\nc film Vision15\nWorking Photoacoustic Electrical Photovoltaic Photovoltaic Ultrasound Optogenetics\nprinciple\nPhysical 56-μm 200-μm pixel 100-μm pixel 100-μm pixel 81-μm Not\nspatial ultrasound diameter size in diameter ultrasound applicable\nresolution field width and 575-μm patients, 20- and 120-μm field width\npitch μm in pitch\nrodents19\nField of view 16.7° 11° × 19° 7° 46.3° 20 Theoretically 8.2°\n> 40°\nPixel density To be tested, 3.6 94.5 79.1 1.8 Not\ntheoretically pixel/mm2 pixel/mm2 in pixel/mm2 21 pixel/mm2 on applicable\n> 2500 patients device\npixel/mm2\nRestored To be tested 20/158822 20/500 in To be tested, To be tested 20/249\nvision acuity patients, theoretically\n20/100 in ~<20/400\nrodents19 based on\nresolution\nStimulation 290 mW/mm 1 mC/cm2 3.5 33 μW/mm2 3 MPa @ 4.4 13 mW/cm2\nintensity mW/mm2 MHz*\nImplant Subretinal Epiretinal Subretinal Epiretinal Noninvasive Intravitreal\nposition space space space space injection\nDevelopment Preclinical in FDA Multicentric Preclinical in Preclinical in Pilot clinical\nstage rodents approved clinical trial minipigs rodents trial\n* Mechanical index exceeds FDA safety requirement in the frequency range <14 MHz and ~18 MHz.\nSupplementary Table T2 A comparative table between our approach and other vision restoration\ntechnologies across multiple parameters.\n13. The author missed the statistical test description for latency differences for the distribution of\nresponse latencies (p. 9, Figure 2g).\nThe statistical test used is Wilxocon rank-sum, and the text was updated accordingly in line 199.\n14. The author used functional ultrasound imaging (fUS) to measure cerebral blood volume (rCBV) in\nthe superior colliculus as an indirect indicator of retinal stimulation. However, are there any baseline\nactivity comparisons for the superior colliculus before and after implant? It makes it difficult to\nseparate true stimulation effects from baseline vascular fluctuations.\nThe rat skull is too thick for fUSI recording without skull thinning or removing the skull above\nthe region of interest (cranial window). All of the fUSI recordings performed in our study were\nperformed with a cranial window, and were acute recordings, i.e. the animal was euthanized directly\nafter the recording. We did not perform any fUSI recording before implantation because of this\nlimitation. This constraint of the experiment was introduced in the result section to clarify our protocol\n(lines 406-407) :\n“A cranial window was generated just prior to the experiment, preventing a long-term follow-up on\nthe colliculus activity.”\nMoreover, it has been shown that RGCs do not degenerate after subretinal implantation12,13.\nTherefore, we do not expect any effects on the brain, nor do we expect the baseline vascular activity\nof an implanted rat to be different from the baseline of a non implanted rat.\nBaseline vascular fluctuations were measured for a duration of 40 s prior to and following each\nlaser stimulation session, which consisted of 15 laser bursts spaced 15 s apart (Fig. 7d). We have added\na sentence for better clarification in the legend of Fig. 7d, in Lines 430-432.\nWe separated the stimulation effects from the baseline vascular fluctuations by normalizing\nthe cerebral blood volume (CBV) into a steady-state value (relative cerebral blood volume, rCBV),\nwhich is calculated as the following: rCBV = (CBV(t) - CBV )/CBV , where CBV(t) is the power doppler\n0 0\nvalue t seconds after the start of laser sequence, and CBV is the average of the baseline activity 5 s\n0\nbefore the start of the stimulation session (see Material and Methods, Lines 725-730). Therefore, any\nsignificant variation in activity shown in the in vivo section is separate from baseline vascular\nfluctuation.\n12. Lorach, H. et al. Development of Animal Models of Local Retinal Degeneration. Investig.\nOpthalmology Vis. Sci. 56, 4644 (2015).\n13. Prévot, P.-H. et al. Behavioural responses to a photovoltaic subretinal prosthesis implanted in\nnon-human primates. Nat. Biomed. Eng. 4, 172–180 (2019).\n15. The author claim a lateral resolution of 56 µm, based on PA field mapping (p. 14, Fig. 4c). However,\nwhen reading this paper, it said the MEA electrode array used for RGC recordings has a 100-µm pitch,\nmeaning that single-cell resolution cannot be accurately confirmed at the stated resolution. Please\nexplain how this is confirmed because the true resolution might be much lower due to the averaging\nof responses across multiple electrodes.\nThe ultrasound produced with our photoacoustic film was mapped through PA-field mapping\n(Fig. 1d), as described in the Material and Methods (Lines 583-591). The setup (we added a new figure,\ncurrent Supplementary Fig. S7) is different from the one used for the ex vivo MEA recordings (Fig. 2a),\nand is better described in Chen et al.14. In simple terms, PA-field mapping is an optical technique for\nmeasuring the acoustic field distribution with high resolution. This allowed us to determine a lateral\nresolution of 56 µm upon illumination with a 50-µm optical fiber. We want to emphasize that this\nlateral resolution is not enough for single-cell resolution, so no such statement was claimed in the\nmanuscript.\nTo avoid further confusion, we have added the following sentences in the Discussion (lines\n508-511):\n“We demonstrated through PA-field mapping that a 50-µm-diameter laser spot generates a 56-µm\nlateral ultrasound field, and that the RGC activity was locally modulated around the laser spot in both\nhealthy and degenerated retinae. Neuronal spatial resolution based on MEA recordings still remains\nto be determined.”\nWe have added schematics of the PA-mapping setup in the Supplementary Information\n(Supplementary Fig. S7), which is also shown below. The Material and Methods have also been\nmodified to cite this figure.\nSupplementary Figure S7. Setup for spatial-offset pump-probe imaging used to acquire the\nphotoacoustic field. Adapted with permission from Chen et al.25. The PDMS/CS/PDMS film was cut\ninto small pieces and attached to the end of the optical fiber for characterization. The nanosecond\npump laser, delivered through an optical fiber, generated the photoacoustic signal. An orthogonally\naligned probe laser detected the resulting ultrasound field by sensing changes in optical signal\namplitude caused by ultrasound-induced density variations in the medium. More details about the\nsetup can be found in Chen et al.25\n14. Chen, G. et al. Spatial-offset pump-probe imaging of nonradiative dynamics at optical\nresolution. Sci. Adv. 11, eadw4939 (2025).\nReviewer #2 (Remarks to the Author):\nThe work from Leong et al. proposes the technological translation to in vivo of a previous\nphotoacoustic neuronal stimulation strategy for vision restoration.\nAlthough the device presents a very low energy conversion efficiency and it does not represent a\nnovelty per se, the work is well structured and suitable for publication upon revision of a few\nimportant issues.\nFirst, the assessment of the toxicity/biocompatibility of the device should not be limited to the\nmeasurement of the retina thickness but include an immuno/histopathological study of the retina (i.e.\nIba1 and GFAP, or CD86…, and photoreceptors markers).\nWe thank the reviewer for their suggestion of adding immunofluorescence staining results.\nWe have done whole-mount immuno-histochemistry assays on 3 LE rats and 4 P23H rats. We stained\nfor microglia (Iba1), astrocytes (GFAP), rods (rhodopsin) and cones (cone arrestin).\nWe have observed the presence of activated microglia at the implantation site in both LE (Fig.\n6e, ii) and P23H rats (Fig. 6f, ii). Activated Müller glia were only present in LE rats (Fig 6e, iii; P23H: Fig\n6f, iv). We observed no significant inflammation (i.e. increase in GFAP expression and in microglia\nnumber or size) surrounding the implant (Fig 6e-f, iv), except for a zone which corresponds to its\ninsertion track (Fig. 6e, iv, bottom right corner).\nMoreover, we confirmed the degeneration of photoreceptors at the location of the implant\nin LE rats (Fig. 6e, i) with the loss of both the rhodopsin and the cone-arrestin immunolabelings. This\nobservation is consistent with the retinal thinning at the implant position after 15 dpi (Fig. 6c).\nWe have added the panels presented below to Figure 6 (Fig. 6e-f), added a part on\nimmunohistochemistry in the Material and Methods section, and added Clémence Bradic in the list of\nauthors, for her contribution to the histological results.\nFigure 6. In vivo photoacoustic implant biocompatibility. (e) Immunolabeling of an implanted LE\nretina, labeling rods (Rho, red, i, iv), cones (CAR, green, i, iii), microglia (Iba1, magenta, i, ii, iv), and\nMüller cells (GFAP, green, i, iii). i - iii: implanted zone. iv: zone surrounding the implant. (f) Same as (e)\nfor P23H, with microglia (Iba1, magenta, i, ii, iv) and Müller cells (GFAP, green, i, iii). Scale bar for (e)\nand (f) is 200 µm.\nSecond, the in vivo results, although extremely solid, would be given more relevance by the\nemployment of a dystrophic animal relying on a pathological degeneration of the photoreceptors with\nconsequent rewiring of the inner retina. As mentioned before, although proven for other prosthetic\nsolutions, IF/IHC of the retina below/above the implant should be included to prove the lack of light\nsensitivity.\nWe agree that the use of dystrophic animals would provide valuable insight into the suitability\nof photoacoustic stimulation for vision restoration in blind patients affected by conditions such as\nretinitis pigmentosa. We started this experimental series by implanting dystrophic P23H rats.\nHowever, we did not manage to observe any significant activation of visual structures of the brain\nusing fUSI. The absence of natural visual responses for ensuring the position of the fUSI probe made\nus decide to use LE rats with a restricted degenerated patch at the implant position. As addressed\nabove, the immunohistological examination of the whole-mounted retinae of implanted wild-type\nanimals confirms with these results the complete degeneration of photoreceptors at the location of\nthe implant. Future studies are needed to investigate the feasibility of using fUSI and possible\nalterations of brain neuronal activity in dystrophic animals.\nThird, it is not clear why there is a change in the architecture of the in vivo prosthesis for thickness\npurposes but then both candle soot and CNT prostheses are employed in the experiments for\ncomparison. Please elaborate on the choice of device studied in vivo.\nThe studies with PDMS-CNT and PDMS/CS/PDMS were conducted in parallel. At the time, we\nassumed that there was a risk of increased degeneration of inner retinal layers with the 115µm-thick\nPDMS/CS/PDMS implants compared to the 40-µm-thick PDMS-CNT implants. We chose to leave the\ndata obtained with PDMS-CNT films in order to show suitability of thin PDMS-CNT film for in-vivo\nimplantation.\nWe have added the following sentence for better clarification, in lines 341-343 of the results\nsection:\n“To investigate whether there is a risk of increased degeneration of inner retinal layers with\nthe 115-µm-thick PDMS/CS/PDMS film, we also prepared and studied a thinner PDMS-CNT film.”\nMinor comments\n1. Figure 1b could be replaced by a SEM image better detailing the device. A top view could be also\nappreciated to immediately get the size of the prosthesis. Same thing for Supplementary Figure S4a.\nAn optical image can characterize the dimensions of the layers in the PDMS/CS/PDMS film.\nWe believe that there is no feature in the film that would require the resolution of a SEM.\nIt is also important to note that the prosthesis is cut from a bigger PDMS/CS/PDMS or PDMS-\nCNT film, using a 1-mm biopsy punch. This size was chosen for ease of implantation in rats, but any\nsize of biopsy punch can be chosen as long as they fit on the film. For instance, the film was cut with\na 1.5-mm biopsy punch for the ex vivo experiments, in order to cover the whole surface of the MEA.\nSince the size of the prosthesis can be modified depending on the application, we don’t think a top\nview picture of a specific size will be representative.\n2. Figure 2i could benefit from a 2-way statistical analysis of variance.\nIndeed, analyses with multiple groups would benefit from an omnibus test like ANOVA.\nBecause we have paired, non-normal distributed data, we opted to use the Friedmanchi square test\nrather than the ANOVA test. The data from previous figure 2i is now presented in Figure 3e, we have\napplied these statistical tests to panels 3b and 3c as well. Finally we applied a correction for multiple\ncomparisons as well.\nWe now describe the statistical test used in the legend of Figure 3:\n“For each panel, groups are tested for significant differences using Friedman chi square, with post-hoc\nWilcoxon signed-rank. p-values are holm-corrected (***: p<0.001, **: p<0.05)“\n3. The title states “high precision” but indeed it is among photoacoustic ones, not overall. Might be\nmisleading?\nWe are aware of the work that provides a spatial resolution of 28 μm in rats15, but we still consider 50\nµm to be high precision. Still, to avoid misleading the reader, we have removed “high precision” from\nour title.\n15. Wang, B.-Y. et al. Electronic photoreceptors enable prosthetic visual acuity matching the\nnatural resolution in rats. Nat. Commun. 13, 6627 (2022).\nReviewer #3 (Remarks to the Author):\nIn their manuscript, the authors describe a novel ultrasound-based retinal stimulation technology.\nThey demonstrate that, by implanting a carbon-core film covered with polymethylsiloxane into the\nsubretinal space in the rat eye, it is possible to generate photoacoustic waves inside the eye by\nstimulating the film with near-infrared light pulses, and that this mechanical stimulation can activate\nretinal neurons and downstream visual pathways in vivo. The topic of the study is very relevant, and\nthe results achieved are of high interest, because they might open up a strategy for creating high-\nprecision retinal prostheses with a wide field of view. The manuscript should be published after some\nrevisions addressed below.\nThe manuscript is thoroughly thought and carefully written, particularly in the demonstration of\nphotoacoustic modulation of the retina, both ex vivo and in vivo, as well as in healthy and\ndegenerated retinas (without photoreceptor cells). However, the safety issues may be a bit less well\naddressed. My main concern is the photoacoustic waves potentially propagating towards the\nchoroid from the film. As I understand it, the two sides of the films surrounding the carbon\nnanoparticle (and nanotube) layer were quite similar, suggesting that the photoacoustic waves\ngenerated by the rapid expansions of the film are likely to propagate in both directions, toward the\nretina on one side and toward the retinal pigment epithelium (RPE) and the choroid on the other\nside. Since the neuronal stimulation is presumed to occur through the activation of\nmechanosensitive channels in the retinal neurons, and RPE cells and the smooth muscles in\nvasculature contain several types of mechanosensitive channels as well, a discussion on the\npotential effects on the RPE and the choroid should be added in the Discussion section. Additionally, if\nnecessary, what approaches could be used to dampen the photoacoustic waves towards\nRPE/choroid? The discussion should also cover the potential effects of photoacoustic stimulation on\nretinal blood vessels.\nWe thank the reviewer for their positive comments, and for the concern raised regarding the\nsafety of ultrasound exposure to the choroid. We want to emphasize that the ultrasound produced\nwith our photoacoustic film falls within the FDA Marketing Clearance of Diagnostic Ultrasound\nSystems and Transducers16 thresholds for ophthalmic devices. Indeed, as shown in the Supplementary\nTable T1, the upper bounds for mechanical index (MI) and spatial-peak, temporal-average intensity\n(I ) are 0.01 and 0.0011 mW.cm-2, respectively, for the PDMS/CS/PDMS film. This is 23- and about\nSPTA\n1500-fold lower than the thresholds set by the FDA, which are 0.23 for MI and 17 mW.cm-2 for I .\nSPTA\nSimilarly, the upper bounds for the PDMS-CNT film are 0.02 for MI and 0.250 mW.cm-2 for I ,\nSPTA\nwhich is 11.5- and 68-fold lower than the recommended maximum values. We believe that, taking these\narguments into account, the safety issues regarding the choroid, RPE, and blood vessels are minimal.\nWe included the following paragraph in the discussion:\n“Due to the bidirectional nature of our implant, acoustic waves are transmitted not only to the retinal\nside, but also to the opposite side toward the RPE and choroid, potentially impacting\nmechanosensitive elements therein. While the implications of ultrasound waves to the RPE and\nchoroid require further investigation, the applied acoustic pressures are below safety thresholds, and\nthe acoustic waves are applied at 0.025% duty cycle. Thus, limited or no aversive effects are to be\nexpected on the RPE and choroid.” (lines 491-496)\n16. FDA. Marketing Clearance of Diagnostic Ultrasound Systems and Transducers - Guidance for\nIndustry and Food and Drug Administration Staff. (2023).\nAnother issue to address in Discussion is the question, how well mechanosensitivity is preserved in\nthe retina. Could continuous “visual” mechanostimulation lead to some form of adaptation or\ndepression of retinal mechanosensitivity? This information would be highly relevant for the potential\ndevelopment of photoacoustic retinal prostheses.\nTo the best of our knowledge, there have been no published studies addressing the long-term\neffects of retinal mechanostimulation for the purpose of vision restoration. Since the structures\ninvolved in retinal mechanosensitivity are not yet known, it is difficult to evaluate the potential\nadaptation or depression of mechanosensitivity.\nIt has been shown that retinal stretch with increased intraocular pressure (IOP) resulted in an\nincreased expression of Piezo1 and 2 in RGCs17. While the similarities between the mechanisms\nunderlying elevated IOP and \"visual\" mechanostimulation are not yet understood, it is conceivable\nthat \"visual\" mechanostimulation may also lead to an increase in the expression of mechanosensitive\nproteins. In the course of our studies, no such depression was observed, but the stimulations were\nlimited in time. Future studies need to be conducted to better evaluate the effect of long-term\nmechanical stimulation of the retina.\nWe have added a paragraph in the Discussion (lines 517-525) to address this concern:\n“Since the structures involved in retinal mechanosensitivity are not yet known, it is difficult to evaluate\nthe potential adaptation or depression of mechanosensitivity to long-term ultrasonic retinal\nstimulation for vision restoration. It has been shown that retinal stretch with increased intraocular\npressure (IOP) resulted in an increased expression of Piezo1 and 2 in RGCs25. While the similarities\nbetween the mechanisms underlying elevated IOP and ultrasonic mechanostimulation are not yet\nunderstood, it is conceivable that ultrasonic mechanostimulation may also lead to an increase in the\nexpression of mechanosensitive proteins. In the course of our studies, no such depression was\nobserved, but the stimulations were limited in time. Future studies need to be conducted to better\nevaluate the effect of long-term mechanical stimulation of the retina. ”\n17. Sripinun, P. et al. Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1\nstimulation on light-dependent neural activity. 2024.06.25.599602 Preprint at\nhttps://doi.org/10.1101/2024.06.25.599602 (2024).\nSome other points to be considered:\nFig. 1g: The authors write that they measured the temperature “at the surface of the film”. With the\nrobust size of the thermocouple (diameter 200 µm), especially when compared to the thickness of the\nretinas, the temperature values given may rather describe those in the near surroundings of the film.\nIndeed, what we measured is the temperature at the surface of the film through a direct\ncontact between the photoacoustic film upon laser illumination and the thermal couple. Specifically,\na small piece of PDMS/CS/PDMS film was placed on the tip of a 200-µm optic fiber, which was\nsubsequently aligned with the 200-µm thermocouple to ensure direct contact between the\nthermocouple and the film. The whole setup was embedded in agarose to maintain the alignment\nduring the measurements, as shown in Supplementary Figure S5 and described in the Material and\nMethods (Lines 592-600).\nIn addition, as detailed in our response regarding the safety issues for the choroid and\nsurrounding ocular tissues, photoacoustic neuromodulation generates only a single ultrasound pulse\nwith a duty cycle of 0.025%. This indicates that heat accumulation from ultrasound exposure is\nminimal, contrary to conventional ultrasound neuromodulation. Instead, the primary source of heat\nin the photoacoustic process is laser absorption at the CS layer, which is more dependent on laser\ndosage than ultrasound frequency. In our case, as indicated in line 109, we only observe an average\nheating of 0.52 ± 0.09 °C with our stimulation parameters. Taking this into account, we expect the\nhighest temperature increase for the surrounding tissue to occur at the surface of the film.\nTo answer this concern, we added the following sentences to introduce the measurement\n(lines 103-105):\n“The greatest heat increase is assumed to be generated at the level of the PA film, as a consequence\nof the laser light absorption. Ultrasound absorption could cause an additional increase in temperature,\nbut it is expected to be much lower due to the very low acoustic energy.”\nThe authors suggest on line 154 that photoreceptor cells may partly contribute to the ex vivo retinal\nmechanosensitivity. While it is not very essential for the study, blocking synaptic transmission from\nphotoreceptor cells to bipolar cells using L-AP-4 would have provided a better comparison of the\neffectiveness of photoacoustic stimulation between the LE and P23H retinas by revealing the\ncontribution of photoreceptors. Did the authors test the effect of L-AP-4 on mechanosensitivity in LE\nretinas?\nWe thank the reviewer for their suggestion of experiment. We have performed additional\nexperiments to test the contribution of photoreceptors to retinal mechanosensitivity by bath-applying\nL-AP4 and ACET to ex vivo LE retinae (n = 2 retinae, 181 recorded cells, 101 PA-responsive cells before\nblockers). ACET is a kainate antagonist which was used to block remaining PA responses after L-AP4\napplication.\nFor clarity’s sake, we have modified the structure of our manuscript to add a section on\nsynaptic transmission blocking. An additional experiment has been done with (rs)-CPP and CNQX with\na P23H retina in order to get a better significance of the results. Current Figure 3d-e takes into account\nthis new experiment. The results of the L-AP4 experiments are shown in Figure 3a-c. The new Figure\n3 (page 13-14) is also shown below. The Material and Methods were also modified to include the use\nof L-AP4 and ACET, in lines 627-631.\nApplication of L-AP4 to LE retinae induced a reduction in the fraction of PA-responsive cells\n(19% vs 58% before L-AP4, Fig. 3b, L-AP4 and No blocker) as well as a decrease in response firing rate\n(from 103 ± 2 Hz to 37 ± 6 Hz, Fig. 3b, No blocker and L-AP4). Interestingly, L-AP4 blocked the fast\nresponses (<45 ms, Fig. 3c, L-AP4), with 55% of responsive cells having a fast response before blockers\nto only 7% after L-AP4. The shift in firing rate post-stimulation and in response latency is similar to\nwhat we observed with P23H RGCs (p = 0.993, 2-sided Mann Whitney-U). Taken together, these results\ndemonstrate the important contribution of photoreceptors in the short-latency response to\nultrasound stimulation\nThe following text was added to the results section, in lines 215-237:\n“Mechanistic exploration of retinal mechanosensitivity\nIt is currently unclear which layers in the retina contribute to the mechanosensitivity of the retina. We\nfirst observed an increase in response latency when photoreceptors have degenerated in P23H rat\nretinae, compared to LE retinae (Fig. 2g). To assess if photoreceptors initiate the short-latency US\nresponse, we bath-applied the group III mGluR agonist L-AP4 to LE retinae (Fig. 3a-c), which blocks the\nsynaptic transmission between photoreceptors and ON bipolar cells43. We subsequently applied the\nkainate agonist ACET, which blocks most of the remaining glutamatergic synapses.\nBefore application of the blockers, a large fraction of recorded RGCs was responsive to the PA\nstimulation (58%, Fig. 3a, No blocker) with strong response firing rates (103 ± 2 Hz, Fig. 3a, No blocker).\nFollowing L-AP4 application, fewer RGCs were responsive to PA stimulation (19%, Fig. 3b, L-AP4), and\ntheir firing rates following stimulation were highly decreased (37 ± 6 Hz, Fig. 3b, L-AP4). Moreover, L-\nAP4 predominantly suppressed short-latency responses (<45 ms, 7% of recorded cells, Fig. 3c, L-AP4).\nThese short-latency responses were recovered after the washout of the L-AP4 (Fig. 3c, baseline and\nwashout): The L-AP4 condition mimics what was observed with recordings of P23H rat retinae with\ndegenerated photoreceptors (p = 0.993, 2-sided Mann Whitney-U). These results further support that\nphotoreceptors generate most of the short-latency RGC responses to ultrasound.\nAddition of ACET to L-AP4 further suppressed RGC responses to PA stimulation (L-AP4 + ACET: 8 %, L-\nAP4: 19%, control with no blockers; 55%, Fig. 3b). This additional effect of ACET indicated that cells in\nthe inner retina, upstream of RGCs, are also contributing to the production of the US-mediated RGC\nactivation. These pharmacological data indicate that part of the RGC responses to PA stimulation rely\non upstream signaling pathways. Taken together, these results demonstrate the important\ncontribution of photoreceptors in the short-latency response to ultrasound stimulation, as shown by\nprevious studies6,18, and reliance of PA-mediated stimulation on upstream signaling.”\nFigure 3. Photoacoustic responses under pharmacological blocking. (a) Percentage of cells\nmodulated by photoacoustic stimulation per stimulation site before and after L-AP4 application. No\nblocker: 49%; L-AP4: 16%; L-AP4 + ACET: 7%; washout: 40% (2 retinae, n = 9 stimulation sites). (b)\nGroup III mGluR agonist L-AP4 decreased RGC spontaneous firing rate and firing rate during PA-elicited\nresponses in LE retinae. The population firing rate of RGCs (n = 41 cells, 2 retinae) is compared between\nbaseline (Basal) and stimulation (Stim.), before blocker application (No blocker), following blocker\napplication (L-AP4) and after washout (Washout) with Ringer medium. Firing rates per condition, no\nblockers: frbasal = 18 ± 2Hz, frstim = 103 ± 9 Hz (p < 0.001); L-AP4: frbasal = 9 ± 1 Hz, frstim = 37 ± 6 Hz (p\n< 0.001); L-AP4+ACET: frbasal = 6 ± 1 Hz, frstim = 16 ± 3 Hz (p = 0.024), washout: frbasal = 7 ± 1 Hz, frstim\n= 86 ± 13 Hz (p < 0.001). Comparison of the population stimulation firing rate frstim after blocker\napplication and frstim before application (p < 0.001), and frstim after washout (p < 0.001). (c) L-AP4\nincreases response latency in LE retinae. Response latency per condition, no blockers: 49 ± 5 ms; L-\nAP4: 65 ± 4 ms; washout: 42 ± 3 ms. Comparison of the response latency before blocker application\nand after (p < 0.001, Wilcoxon signed-rank), and after washout (Friedmanchi square, p = 0.4). Due to\nabsence of responses during application of L-AP4+ACET blockers, no latencies are reported for this\ngroup. Dashed black line: 45-ms cutoff for slow- and fast-latency responses. (d) Percentage of cells\nmodulated by photoacoustic stimulation per stimulation site before and after (rs)-CPP and CNQX\napplication. No blocker: 42 %; (rs)-CPP + CNQX: 5 %; washout: 8 % (2 retinae, n = 5 stimulation sites).\n(e) Glutamate blockers (rs)-CPP and CNQX abolished RGC responses to PA stimulation in P23H retinae.\nThe population firing rate of RGCs (n = 25 cells, 2 retinae) is compared between baseline (basal) and\nstimulation (stim), before blocker application (no blocker), following blocker application ((rs)-\nCPP+CNQX) and after washout (washout) with Ringer medium. Firing rates per condition, no blockers:\nfrbasal = 11 ± 2 Hz, frstim = 100 ± 23 Hz (p < 0.001); (rs)-CPP+CNQX: frbasal = 7 ± 2 Hz, frStim = 23 ± 10 Hz\n(p =0.141); washout: frbasal = 8 ± 2 Hz, frstim = 24 ± 9 Hz (p = 0.016). Comparison of the population\nstimulation firing rate fr after blocker application and fr before application (p < 0.001, Wilcoxon\nstim stim\nsigned-rank), and frstim after washout (p < 0.001, Wilcoxon signed-rank). For each panel, groups are\ntested for significant differences using Friedman chi square, with post-hoc Wilcoxon signed-rank. p-\nvalues are holm-corrected (***: p<0.001, **: p<0.05)\nFig. 2 legend: (a) replace “on the outer layer” with “on the photoreceptor layer”\nWe modified the sentence in the Figure 2 legend, page 10, lines 180-181.\nLine 217: replace “requires higher thresholds for stimulation” with “requires stronger stimulation”\nWe modified the sentence in the results section, line 284.\nLines 274-275: subretinal space might be mentioned\nWe modified the sentence to “To test the feasibility of photoacoustic stimulation and the\nbiosafety of the film in vivo, we chronically implanted 1-mm-diameter PA films in the subretinal space\nof LE and P23H rats.”, line 340.\nLines 323-324: Were the pupils dilated during full-field white light stimulations?\nThe pupil of the implanted eye was dilated with a tropicamide-based solution before full-field\nwhite light stimulation, as indicated in the Material and Methods (lines 719-720), and was reapplied\nas needed for any subsequent stimulation.\nLine 387: The authors mention that “we developed flexible photoacoustic films...”. While true,\nsimilar PDMS-CS-PDMS films have been developed earlier, e.g. Chang et al.: Candle soot\nnanoparticles-polydimethylsiloxane composites for laser ultrasound transducers. Appl. Phys. Lett.\n107, 161903 (2015). References to prior works should be added, also in Material and methods.\nPDMS- and CS-composite-based films have indeed been developed for applications in\nphotoacoustic generation and other related fields. We have added citations in both the Discussion\n(lines 474-478) and Materials and Methods (lines 540-542) sections, as suggested:\n“Although CS-PDMS films have been developed as laser ultrasound transducers23–25 and for\nsuperhydrophobic coating26,27, our study represents the first instance of engineering them into a\nsandwich-structured PDMS-CS-PDMS film as a biomedical implant, specifically designed to minimize\nheat deposition in the surrounding tissue and to minimize the diffusion of carbon particles into the\nbiological tissue.” (Discussion, lines 474-478)\n“Although CS-PDMS films have been developed as laser ultrasound transducers23–25, our study\nrepresents the first instance of engineering them into a sandwich-structure.” (Materials and Methods,\nlines 540-542)\nIt is important to highlight that our film represents a novel design characterized by a sandwich\nstructure, where pure PDMS is in direct contact with the biological tissue. This configuration,\nspecifically engineered for biomedical implantation, aims to reduce the risk of carbon particle leaching\ninto the eye, and to minimize thermal accumulation in the surrounding tissue. Importantly, the\npresence of a pure PDMS layer exceeding 10 µm in thickness between the absorber and the biological\ntissue effectively prevents transient temperature elevations resulting from individual laser pulses\n(Supplementary Information, section 3.2).\nLine 514: 5-nm instead of 0.5-nm\nWe modified the sentence in the Material and Methods, in line 638.\nReferences\n1. Shi, L. et al. Non-genetic photoacoustic stimulation of single neurons by a tapered\nfiber optoacoustic emitter. Light Sci. Appl. 10, 143 (2021).\n2. Menz, M. D. et al. Radiation Force as a Physical Mechanism for Ultrasonic\nNeurostimulation of the Ex Vivo Retina. J. Neurosci. 39, 6251–6264 (2019).\n3. Thoreson, W. B. & Ulphani, J. S. Pharmacology of selective and non-selective\nmetabotropic glutamate receptor agonists at L-AP4 receptors in retinal ON bipolar cells. Brain Res.\n676, 93–102 (1995).\n4. Khabou, H. et al. Optogenetic targeting of AII amacrine cells restores retinal\ncomputations performed by the inner retina. Mol. Ther. Methods Clin. Dev. 31, (2023).\n5. Pisauro, M. A., Dhruv, N. T., Carandini, M. & Benucci, A. Fast Hemodynamic Responses\nin the Visual Cortex of the Awake Mouse. J. Neurosci. 33, 18343–18351 (2013).\n6. Provansal, M. et al. Functional ultrasound imaging of the spreading activity following\noptogenetic stimulation of the rat visual cortex. Sci. Rep. 11, 12603 (2021).\n7. Ellis, E. M., Gauvain, G., Sivyer, B. & Murphy, G. J. Shared and distinct retinal input to\nthe mouse superior colliculus and dorsal lateral geniculate nucleus. J. Neurophysiol. 116, 602–610\n(2016).\n8. Seabrook, T. A., Burbridge, T. J., Crair, M. C. & Huberman, A. D. Architecture, Function,\nand Assembly of the Mouse Visual System. Annu. Rev. Neurosci. 40, 499–538 (2017).\n9. Duangkanya, K., Kopwitthaya, A., Chanhorm, S. & Infahsaeng, Y. Oxygen plasma\ntreatment time induced hydrophilicity of polydimethylsiloxane (PDMS) thin films for liquid lenses\napplication. Mater. Today Proc. 65, 2442–2445 (2022).\n10. Chuah, Y. J. et al. Surface modifications to polydimethylsiloxane substrate for stabilizing\nprolonged bone marrow stromal cell culture. Colloids Surf. B Biointerfaces 191, 110995 (2020).\n11. Jastrzebska, E. et al. Biological characterization of the modified poly(dimethylsiloxane)\nsurfaces based on cell attachment and toxicity assays. Biomicrofluidics 12, 044105 (2018).\n12. Lorach, H. et al. Development of Animal Models of Local Retinal Degeneration. Investig.\nOpthalmology Vis. Sci. 56, 4644 (2015).\n13. Prévot, P.-H. et al. Behavioural responses to a photovoltaic subretinal prosthesis implanted in\nnon-human primates. Nat. Biomed. Eng. 4, 172–180 (2019).\n14. Chen, G. et al. Spatial-offset pump-probe imaging of nonradiative dynamics at optical\nresolution. Sci. Adv. 11, eadw4939 (2025).\n15. Wang, B.-Y. et al. Electronic photoreceptors enable prosthetic visual acuity matching the\nnatural resolution in rats. Nat. Commun. 13, 6627 (2022).\n16. FDA. Marketing Clearance of Diagnostic Ultrasound Systems and Transducers - Guidance for\nIndustry and Food and Drug Administration Staff. (2023).\n17. Sripinun, P. et al. Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1\nstimulation on light-dependent neural activity. 2024.06.25.599602 Preprint at\nhttps://doi.org/10.1101/2024.06.25.599602 (2024)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author has revised the manuscript reviewers asked. Before publication, could you please add some minor concerns in the discussion part? (1) How laser beam can focus on the 1 mm thin films for photoacoustic signal? (2) using one fiber, how to make beam pattern for retinal prothesis?", "reviewer_2": "The authors have addressed my comments constructively and satisfactorily. I therefore have no further objections to publication.", "reviewer_3": "The manuscript has been improved and most of my questions have been answered.\nHowever, I still remain concerned about the acoustic stimulation of the RPE, the key cells in the blood-retina barrier. The distance from the photoacoustic film to the RPE is either smaller than or comparable to that to the retinal ganglion cells. Since RPE cells are known to contain mechanosensitive channels (Piezo 1 and 2, as well as others), photoacoustic stimulation might, for example, persistently elevate the intracellular calcium concentrations in RPE cells, thereby affecting the connectivity between RPE cells as well as other calcium-sensitive mechanisms in the RPE. Therefore, I feel that considering only acoustic pressure as the source of potential adverse effects is inadequate, and a brief comment on possible effects through the mechanosensitive mechanisms in the RPE should be added to the Discussion."}, "author_response": "General statement\nWe would like to thank the reviewers again for their constructive feedback and remarks.\nWe have tried to rigorously address the additional concerns of reviewers 1 and 3 by adding\nmore details in the Discussion section.\nWe have noticed a mistake concerning the lateral width measurement of the ultrasound field\nproduced by our PDMS/CS/PDMS film, presented in Figure 1e. We apologize for this mistake, and have\nappropriately corrected Figure 1e, its legend, as well as the corresponding text in the manuscript. This\nexplains why the lateral resolution is now 51 µm, instead of the 56 µm initially claimed.\nTo comply with Nature Communications submission guidelines, the legends of the figures 1, 2,\nand 3 were shortened. Namely, most of the statistics were moved to the main text. When applicable,\nmethodological descriptions were removed, and adequately added to the Methods section if they were\nnot already present. Figures 2h and 2i were displaced to Supplementary Figure S9. We have also moved\nall of the figure legends to the end of the manuscript, and submitted the figures as separate files.\nAs before, all of the changes have been highlighted in red.\nResponse to reviewer comments\nReviewer #1 (Remarks to the Author):\nThe author has revised the manuscript reviewers asked. Before publication, could you please add some\nminor concerns in the discussion part? (1) How laser beam can focus on the 1 mm thin films for\nphotoacoustic signal? (2) using one fiber, how to make beam pattern for retinal prothesis?\n(1) To deliver a focused laser spot through an optical pathway, a customized projection system can be\ndesigned. For instance, a 4f optical configuration can be used to adjust the magnification of the laser\npattern, ensuring accurate delivery to the retinal plane. By selecting appropriate lens focal lengths, the\nprojected pattern can be precisely focused onto the thin photoacoustic film and confined within the 1-\nmm implant area. We added a sentence to address this concern, line 554-555:\n“A customized 4f projection system will be developed to properly focus the pattern in the future tests”.\n(2) The single-core optical fiber was specifically used for single point stimulation. While this setup was\nsufficient to validate that photoacoustic retinal stimulation could elicit responses both ex vivo and in\nvivo, its spatial resolution is limited. Consequently, it cannot produce patterned illumination needed for\ncomplex visual restoration. For future retinal prosthesis applications, patterned illumination could be\nachieved by using a digital micromirror device (DMD), which would then be coupled to a multicore fiber.\nWe have slightly modified the discussion to address this concern, lines 545-546:\n“the pixel number could exceed a million by using digital micromirror devices coupled to a multi-\ncore optic fiber in order to project a laser pattern onto the continuous film.”\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my comments constructively and satisfactorily. I therefore have no further\nobjections to publication.\nReviewer #3 (Remarks to the Author):\nThe manuscript has been improved and most of my questions have been answered.\nHowever, I still remain concerned about the acoustic stimulation of the RPE, the key cells in the blood-\nretina barrier. The distance from the photoacoustic film to the RPE is either smaller than or comparable\nto that to the retinal ganglion cells. Since RPE cells are known to contain mechanosensitive channels\n(Piezo 1 and 2, as well as others), photoacoustic stimulation might, for example, persistently elevate the\nintracellular calcium concentrations in RPE cells, thereby affecting the connectivity between RPE cells as\nwell as other calcium-sensitive mechanisms in the RPE. Therefore, I feel that considering only acoustic\npressure as the source of potential adverse effects is inadequate, and a brief comment on possible\neffects through the mechanosensitive mechanisms in the RPE should be added to the Discussion.\nWe have added the following sentences to the Discussion, to address the reviewer’s concern about\nmechanosensitive mechanisms in the RPE, lines 613-621:\n“Due to the bidirectional nature of our implant, acoustic waves are transmitted not only to the\nretinal side, but also to the opposite side toward the RPE and choroid, potentially impacting\nmechanosensitive elements therein. The implications of ultrasound waves to the RPE and choroid\nrequire further investigation. RPE cells express several mechanosensitive channels, including Piezo1 and\n21, TRPV1, 2 and 42, among others2, which may mediate potential adverse effects. Ultrasound\nstimulation might promote angiogenesis3, which could contribute to diseases such as AMD and diabetic\nretinopathy. It could also trigger calcium concentration changes in RPE cells, which could affect\nconnectivity between RPE cells4 or have a protective action5. While prolonged mechanical stretch might\ninduce apoptotic signaling, RPE cells were shown to recover following stretch withdrawal6. Since the\napplied acoustic pressures are below safety thresholds and the acoustic waves are applied at 0.025%\nduty cycle, limited or no aversive effects are to be expected on the RPE and choroid. Future studies\nbeyond the scope of this paper will have to address this safety issue of the in vivo RPE ultrasound\nstimulation.”\n1. Sripinun, P. et al. Piezo1 and Piezo2 channels in retinal ganglion cells and the impact of Piezo1\nstimulation on light-dependent neural activity. 2024.06.25.599602 Preprint at\nhttps://doi.org/10.1101/2024.06.25.599602 (2024).\n2. Pang, J.-J. The Variety of Mechanosensitive Ion Channels in Retinal Neurons. Int. J. Mol. Sci. 25, 4877\n(2024).\n3. Ashimori, A. et al. HIF-1α-dependent upregulation of angiogenic factors by mechanical stimulation in\nretinal pigment epithelial cells. Dis. Model. Mech. 17, dmm050640 (2024).\n4. Pearson, R. A. et al. Ca(2+) signalling and gap junction coupling within and between pigment\nepithelium and neural retina in the developing chick. Eur. J. Neurosci. 19, 2435–2445 (2004).\n5. Sheu, S.-J., Wu, S.-N. & Hu, D.-N. Stretch-Stimulated Activity of Large Conductance Calcium-Activated\nPotassium Channels in Human Retinal Pigment Epithelial Cells. J. Ocul. Pharmacol. Ther. 21, 429–435\n(2005).\n6. Wu, S. et al. Cyclic stretch induced-retinal pigment epithelial cell apoptosis and cytokine changes.\nBMC Ophthalmol. 17, 208 (2017)."}]

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