Fabrication of Au nanoparticle-decorated TiO 2 NW arrays

The oriented Au–TiO 2 NW arrays were grown on fluorine-doped tin oxide (FTO, Supplementary Fig. 1) or flexible polymer substrates (Supplementary Fig. 2) by a hydrothermal method26,27, followed by in-situ reduction of gold salt on the surface of pre-formed TiO 2 NW arrays (Methods). The obtained Au-TiO 2 NW arrays had a perpendicular orientation to the underlying substrate, and the average NW diameter and length were ~100 nm and ~2 μm, respectively (Fig. 2a, b), similar to those of pristine TiO 2 NW arrays before deposition of Au nanoparticles (Supplementary Fig. 1a, b). The density of NWs was ~10 per μm2, corresponding to ~109 NW arrays per cm2. X-ray diffraction (XRD) patterns of the Au-TiO 2 NW arrays displayed characteristic reflection peaks of a rutile TiO 2 crystalline structure (Joint Committee on Powder Diffraction Standards, JCPDS No. 21-1276) and a face-center-cubic Au structure (JCPDS No. 04-0784) (Fig. 2c)28. Transmission electron microscopy (TEM) images showed the average size of surface-decorated Au nanoparticles was ~10 ± 5 nm, with the inter-particle distance ranging from 10 to 30 nm (Fig. 2d). High-resolution TEM (HRTEM) images (Fig. 2e, Supplementary Fig. 1c) and the selected-area electron diffraction (SAED) patterns (inset in Supplementary Fig. 1c) revealed the single-crystalline nature of both the NW trunks and the surface-decorated Au nanoparticles, respectively. The well-resolved lattice fringes of 0.324 and 0.248 nm (Fig. 2e) were correlated to the d-spacing values of (110) and (101) planes of rutile TiO 2 crystals29. The lattice spacing of 0.235 nm was assigned to the (111) planes of Au crystal with a face-center-cubic structure. Energy-dispersive X-ray (EDX) spectroscopy (Supplementary Fig. 3) and mapping of Ti, O and Au (Supplementary Fig. 4) further confirmed the composition of Au-TiO 2 NW arrays, as well as the uniform distribution of Au nanoparticles over entire TiO 2 NW arrays. The molar percentages of Au and Ti were calculated as ~0.11% and 71.77%, respectively. X-ray photoelectron spectroscopy (XPS, Fig. 2f) revealed two peaks for Ti as 2p 1/2 and 2p 3/2 , and two peaks for Au 4f 5/2 and 4f 7/2 .

Fig. 2 Photovoltaic performances of Au-TiO 2 NW arrays. a Top-view and b side-view scanning electron Mmcroscope images of Au–TiO 2 NW arrays. c XRD spectra collected for Au–TiO 2 NW arrays. The black and blue numbers indicate the diffraction peaks of TiO 2 and Au, respectively. Diffraction peaks of the FTO substrate are marked by asterisks. d TEM and e HRTEM images of a representative Au–TiO 2 NW, where Au nanoparticles were grown on the NW surface. Inset in d: schematic of the Au–TiO 2 NW structure. f XPS spectra of Au–TiO 2 NW arrays. Top: Ti 2p 1/2 , and 2p 3/2 peaks. Down: Au 4f 5/2 and 4f 7/2 peaks. g UV-visible absorbance spectra of TiO 2 and Au-TiO 2 NW arrays on FTO substrates. h Histogram of photocurrents from Au–TiO 2 NW arrays by illumination of UV, blue and green light, respectively (n = 3). Different filters were applied to obtain near UV (375/28 nm, full intensity 133 μW mm–2), blue (470/20 nm, full intensity 691 μW mm–2), and green light (546/12 nm, full intensity 470 μW mm–2). Data are presented as mean and standard error of mean (S.E.M.). Scale bars: 500 nm (a), 2 μm (b), 20 nm (d), 5 nm (e) Full size image

The decoration of Au nanoparticles can enhance the photoconversion efficiency of TiO 2 NW arrays into visible range, due to the electrical field amplification and the injection of surface plasmon resonance generated hot electrons into TiO 2 conduction band30. The photoabsorption property of Au–TiO 2 NW arrays was first demonstrated by UV-visible absorption spectroscopy (Fig. 2g). Compared to pristine TiO 2 NW arrays that mainly absorb at the UV region, the Au–TiO 2 NW arrays show a much enhanced photoabsorption in the visible range, with the peak position centered around 550 nm, in accordance with the surface plasmon absorption of ~10 nm Au nanoparticles31,32. The absorption coefficients for Au–TiO 2 NW arrays were calculated to be ~1.5, 0.4, and 0.5 μm–1 in the UV, blue and green regimes, respectively, comparable or better than the absorption coefficients of natural photoreceptors (i.e., 0.02–0.06 μm–1 in the green regime)33. These enhanced absorption coefficients are beneficial for Au–TiO 2 NW arrays with a much smaller thickness than normal photoreceptor cell layers, typically on the order of 60 μm.

The photoconversion properties of Au–TiO 2 NW arrays were then demonstrated by photocurrent measurements under different wavelengths of near UV, blue and green light, respectively (Supplementary Fig. 5, Methods). The typical substrate size was ~1 cm2. Compared to the dark state without light, when the Au–TiO 2 NW arrays were illuminated by different light wavelengths, a clear increase of photocurrent was recorded almost instantaneously, while the shutoff of light led to the drop of photocurrent to the original dark state. The summarized photocurrent response of different light wavelengths show the average magnitudes of photocurrent change were ~1450, 108, and 87 pA, respectively (Fig. 2h).

Light responses and spatial resolution

Mice are naturally sensitive to both near UV and visible light. In the following electrophysiology studies, light responses of retinal ganglion cells (RGCs) in wild-type (C57BL/6J) and rd1/cDTA mouse retinas with NW array interfaces were investigated. Mutations in both Pde6b and cDTA genes in rd1/cDTA mice (also referred to as blind mice below) led to complete degeneration of rod and cone photoreceptors by P50 (Fig. 3a, Methods34). To eliminate the possibility of some remaining photoreceptors, we conducted more experiments to measure light responses of RGCs in rd1/cDTA blind retinas. 0/8 RGCs from 4 retinas, 0/7 RGCs from 5 retinas and 0/6 RGCs from 5 retinas responded to near UV (375/28 nm), blue (470/20 nm) and green (546/12 nm) light, respectively, indicating that both rod and cone photoreceptors were completely absent in the rd1/cDTA blind retina. Au-TiO 2 NW arrays were then placed underneath the blind retina, with the inner nuclear layer in contact with NW arrays, and the activities of RGCs were recorded using patch clamp pipettes. The retina and NW arrays were in close contact, as shown in scanning electron microscope (SEM) images (Fig. 3b). Contours of individual retinal cells were not visible in SEM images, as the membranes of retinal cells were embedded in the retinal tissue (Supplementary Fig. 6).

Fig. 3 Light responses in NW arrays-interfaced blind mouse retinas. a Fluorescent images of retinal slices of wild-type and blind mice immunostaining for ChAT (red) and DAPI (blue). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer (photoreceptors). b SEM images of the interface between the retina and NW arrays. c Light responses recorded by whole-cell patch clamp to near UV light stimulation at 0.24 s duration and 10 s (top panel), 5 s (middle panel), 2 s (bottom panel) intervals in NW arrays-interfaced blind retinas. d Percentage of light-responsive cells at different light intensities. Wild-type: 133 μW mm–2, n = 8 RGCs, 6 retinas; 66.5 μW mm–2, n = 8 RGCs, 7 retinas; 33.2 μW mm–2, n = 8 RGCs, 7 retinas; 16.6 μW mm–2, n = 7 RGCs, 5 retinas. Blind + NW: 133 μW mm–2, n = 91 RGCs, 30 retinas; 66.5 μW mm–2, n = 51 RGCs, 19 retinas; 33.2 μW mm–2, n = 47 RGCs, 17 retinas; 16.6 μW mm–2, n = 43 RGCs, 17 retinas (Chi-square test, P 133 = 0.859, P 66.5 = 0.276, P 33.2 = 0.095, P 16.6 = 0.295). e Percentage of light pulses triggering spiking activities in all responsive RGCs. Wild-type: 133 μW mm–2, n = 7 RGCs, 6 retinas; 66.5 μW mm–2, n = 6 RGCs, 5 retinas; 33.2 μW mm–2, n = 6 RGCs, 5 retinas; 16.6 μW mm–2, n = 5 RGCs, 4 retinas. Blind + NW: 133 μW mm–2, n = 65 RGCs, 25 retinas; 66.5 μW mm–2, n = 22 RGCs, 17 retinas; 33.2 μW mm–2, n = 15 RGCs, 11 retinas; 16.6 μW mm–2, n = 18 RGCs, 11 retinas. (Two-sided Wilcoxon rank-sum test, P 133 = 0.212, P 66.5 = 0.815, P 33.2 = 0.913, P 16.6 = 0.974. Data are presented as mean and S.E.M.) f Near UV, blue and green light responses in wild-type, blind and NW arrays-interfaced blind retinas. Horizontal color bars represent the light stimulation color and duration. g RGC responses to weak light illumination in NW arrays-interfaced blind retinas. Scale bars: 20 μm (a), 5 μm (b) Full size image

It is clear that RGCs in the blind retina robustly responded to near UV light (with a 0.24 s duration and intervals of 10, 5 and 2 s, respectively) (Fig. 3c). In addition, to evaluate the light sensitivity of the NW arrays-interfaced blind retina, the spike activities of RGCs in response to light with different intensities were measured (Supplementary Fig. 7a). The intensity-dependent responses to near UV light were similar or close to those in wild-type mice. The fractions of light responsive RGCs in wild-type mouse retinas were similar for different intensities (Fig. 3d, black curve). In the NW arrays-interfaced blind mouse retinas, the fraction of light responsive RGCs were 78%, 47%, 36% and 42% at 133, 66.5, 33.2, and 16.6 μW mm–2, respectively (Fig. 3d, red curve). In addition, RGCs in wild-type mice responded to majority of light pulses (Fig. 3e, black curve). For the NW arrays-interfaced blind mouse retinas, we also showed that the threshold to induce light response was ~0.5 μW mm-2 (Fig. 3g), that was 25 lux, which was out of the intensity range of rods (≤0.1 lux). The percentages of pulses that initiated spike activities in RGCs were similar to those of wild-type retinas (Fig. 3e, red curve). The number of spikes for different light intensities, as well as the spike waveforms were also similar (Supplementary Fig. 7b, g). Due to the absence of photoreceptors and therefore sign-preserving and sign-inverting synapses between the photoreceptors and the bipolar cells, only ON responses were recorded in the NW arrays-interfaced blind mouse retinas. The latency of RGCs in NW arrays-interfaced blind retina was similar to those in wild-type retina at different UV light intensities (Supplementary Fig. 7d). The latencies for green and blue light in NW arrays-interfaced blind retina were much larger than those in wild-type retina, most likely due to the fact that the photocurrent induced by green and blue light was smaller than that by UV light.

The sensitivity of different light wavelengths was then investigated. In wild-type mice, RGCs responded to near UV, blue, and green light (with intensities of 133, 691 and 470 μW mm–2, respectively) (Fig. 3f, top panel). Without the NW arrays, retinas of blind mice did not respond to either UV, blue or green light (Fig. 3f, middle panel), confirming the blindness of these mice. The representative spiking activities of RGCs in response to near UV, blue and green light were shown in Fig. 3f, bottom panel. For each RGC, at least three light pulses were presented, with approximately 1 min interval between each pulse. The average number of elicited spikes was 11 for near UV, 4 for blue and 6 for green light. Light-responsive RGCs responded to 84% near UV light pulses, 57% blue light pulses and 86% green light pulses, respectively (Supplementary Fig. 7c). Moreover, time evolution of RGC responses were measured (Supplementary Fig. 8), indicating that the interface between NW arrays and retinal cells was purely physical contact and did not evolve over time.

In order to examine the anatomical and functional On- and Off-features of RGCs in NW arrays-interfaced blind retina and wild-type retina, we recorded the light responses of RGCs with micropipette filled with internal solutions containing Lucifer Yellow, and then conducted immunostaining of these recorded RGCs to examine the distribution of their dendritic arbors in On and Off layers (labeled by ChAT). We first investigated whether light-sensitive Lucifer Yellow in the pipette affected the light responses in the RGCs35. The percentage of responsive RGCs was similar with or without Lucifer Yellow in the pipette (Supplementary Fig. 9d). In the NW arrays-interfaced blind retina, dendrites of some RGCs were located in both On and Off layers (Fig. 4a, top panel). These RGCs exhibited On responses. Some other RGCs had their dendrites distributed in On layer, and exhibited On responses as well (Fig. 4a, bottom panel). These results indicated that RGCs responded to the onset of light by photocurrent from the NW arrays, despite the fact that they received input from both On-center and Off-center bipolar cells. We further examined the light responsive patterns of RGCs in both NW arrays-interfaced blind retina and wild-type retina. It turns out that the ~30% of the RGCs in NW arrays-interfaced blind retina sustained On responses to light stimulation, whereas the rest RGCs showed transient On responses (Supplementary Fig. 7e). This ratio of transient On to sustained On response was similar to that of wild-type RGCs.

Fig. 4 Anatomical and functional On- and Off- features of RGCs in NW arrays-interfaced blind retina. a Confocal images showing the distribution of dendritic arbors of two example RGCs (cell 1 and 2, left) and their corresponding near UV light responses (right, 133 μW mm–2). White arrows indicate the axon, on-dendritic layer and off-dendritic layer of the Lucifer Yellow labeled RGC. b Responses of two example RGCs to near UV light stimulation (133 μW mm–2) before, during and after the application of glutamate receptor antagonist. Zoom-in spike waveforms are shown to the right of their original figures. Scale bar: 20 μm (a) Full size image

To investigate whether the RGC light responses resulted from direct RGC activation or indirect activation of bipolar cells that induced spiking activities in RGCs, synaptic transmission from bipolar cells to RGCs was blocked using glutamate receptor antagonists (Methods). The light responses from RGCs disappeared 4 min after the application of drugs (Fig. 4b, left and middle columns). After washing off the drugs, the light responses were restored (Fig. 4b, right columns). Light responses of all the RGCs were blocked by the drugs, indicating that light responses in RGCs originate mostly from activation of bipolar cells instead of RGCs (Supplementary Fig. 7h).

The spatial resolution of light response in the NW arrays-interfaced blind mouse retinas was further investigated. The photocurrents induced by light spots were measured with different sizes and different distances from the center of the spot (Supplementary Fig. 9a, b). Near UV and green light spots with different diameters were presented to the RGC (Fig. 5a). For blind mouse retinas, the minimum size of light spots to trigger RGC response was 45 µm. When the diameter of near UV light spot was smaller than 100 µm, 44% of RGCs exhibited clear responses (Fig. 5b). The percentages of RGCs that responded to near UV light spots with diameters of 100–200 µm, 200–300 µm, and 300–600 µm were 79%, 84%, and 96%, respectively. Meanwhile, for green and blue light spots with diameters of 200–300 µm, 31% and 22% of RGCs exhibited light responses, respectively; for green and blue light spots with diameters of 300–600 µm, 36% of RGCs exhibited light responses. In Supplementary Figure 9b, the photocurrent increased significantly when the size of the light spots increased from 200 µm to 300–600 µm, which can lead to the increased percentage of responsive RGCs for the light spots of 300–600 µm. For wild-type mouse retinas, RGCs exhibited a typical center-surround light response pattern for both near UV and green light (Supplementary Fig. 9c).

Fig. 5 Responses of RGCs to light spots of different sizes in NW arrays-interfaced blind mouse retinas. a Left column: near UV light spots with different sizes. Middle column: RGCs (cell bodies and dendritic arborizations) labeled by Lucifer Yellow during whole-cell patch clamp experiments. Right column: responses of RGCs to near UV light spots of different sizes. Purple lines represent the presence of UV light. b Fractions of light-responsive RGCs in NW arrays-interfaced blind mouse retinas that responded to UV, green and blue light spots with different sizes (recordings conducted without Lucifer Yellow in the pipette). UV: 0–100 μm, n = 18 RGCs, 13 retinas; 100–200 μm, n = 19 RGCs, 13 retinas; 200–300 μm, n = 19 RGCs, 13 retinas; 300–600 μm, n = 23 RGCs, 16 retinas. Green: 0–100 μm, n = 13 RGCs, 9 retinas; 100–200 μm, n = 12 RGCs, 9 retinas; 200–300 μm, n = 13 RGCs, 9 retinas; 300–600 μm, n = 25 RGCs, 14 retinas; Blue: 0–100 μm, n = 8 RGCs, 5 retinas; 100–200 μm, n = 9 RGCs, 6 retinas; 200–300 μm, n = 9 RGCs, 6 retinas; 300–600 μm, n = 14 RGCs, 8 retinas. c A sketch map of receptive field calculation of an example neuron. Black circles represent the different locations of light spot. Black dot represents the location of the neuron. d Receptive field diameter of RGCs in NW arrays-interfaced blind mouse retinas. n = 4 RGCs in 4 retinas. Data are presented as mean and S.E.M. Scale bars: 50 μm (a, c) Full size image

In order to estimate the receptive field size of the light-responsive RGCs, we presented light spots of 45 µm in size to the RGCs (Fig. 5c). Since the light spots were manually moved, the light responses were measured at different distances from the cell body of the RGCs along three axes. The responses were roughly homogenous along the three axes. The average size of the receptive field was 316 µm (Fig. 5d). In addition, we moved the light spot of 45 µm across the RGC (Supplementary Fig. 9e). Localized RGC responses were recorded when the light spot swept around the vicinity of the cell body.

Population light responses

In order to demonstrate the capability of the NW arrays-based artificial photoreceptors as potential retinal prosthesis materials, the population light responses in the NW array-interfaced blind mouse retinas using calcium-sensitive protein (GCaMP6s) imaging were also illustrated. 88% of GCaMP6s cells expressed Brn3a, a RGC marker, indicating that majority of the GCaMP6s cells were RGCs (Fig. 6a, b, Supplementary Fig. 10). Light induced responses in both RGCs and underlying neuritis (Fig. 6c, d, Supplementary Movie 1 in the Supporting Information). Some cells had no obvious light-evoked responses (Fig. 6e). The light response curves were similar for all the RGCs (Fig. 6f). 46% RGCs had light responses under whole-field illumination, demonstrating global activation of RGCs. Taken together, these results indicate that the NW arrays-interfaced blind mouse retina has good population light responses.

Fig. 6 Cellular-level population light responses of RGCs in NW arrays-interfaced blind mouse retinas. a Expression of GCaMP6 in the whole-mount retina. Turquoise color represents cell bodies of RGCs that expressed GCaMP6, and magenta color represents Brn3a-labeled RGCs. Arrowhead with long tail: cells co-stained with Brn3a and GCaMP6. Arrowhead with short tail: cells stained with GCaMP6. b The cross section of the retina. INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. c Snapshot image of GCaMP6 fluorescence when no light stimulation was presented. Magenta and blue circles outline 52 light responsive and 66 non-responsive RGCs, respectively. Inset image shows the same snapshot image with no outlines of the RGCs. d All the 52 light responsive RGCs in c. Purple shade represents the presence of near UV light. e Three examples of non-responsive RGCs in c. f Change of fluorescence (ΔF/F 0 ) for all the responsive RGCs in c. Gray lines represent ΔF/F 0 curves for individual cells. Red line represents average ΔF/F 0 for all the RGCs. Purple lines represent the presence of near UV light. Scale bars: 20 μm (a, b), 50 μm (c) Full size image

Light responses of cortical neurons in vivo

To further exhibit the potential utilization of NW arrays in retinal prosthesis, we conducted subretinal implant surgery of NW arrays in blind mice. Figure 7a–c showed the substrate of NW arrays implanted into the subretinal space. 5 months after the implant surgery, the lamination of the blind retina, as well as the RGCs labeled by Brn3a was unaffected by the implant (Fig. 7d). We presented near UV light to one eye of the blind mice and recorded the light-evoked spikes and visually evoked potentials (VEPs) in the contralateral primary visual cortex (Fig. 7e). In the blind mice, no obvious light-evoked spikes were detected (Fig. 7f, left column). UV light elicited robust spiking responses in all implanted blind mice 2 days after implantation (Fig. 7f, middle left column) and wild-type mice (Fig. 7f, right column). In addition, we conducted recordings in chronically implanted mice of both 2 months and 5 months after the implant surgery. The light evoked spiking activities were more prominent in chronically implanted mice (Fig. 7f, middle and middle right columns). UV light also triggered robust local field potentials (LFP) changes in the implanted blind mice (Fig. 7g). The fraction of stimuli that elicited LFP responses for blind mice remained the same for different light intensities, whereas that for the NW arrays-implanted blind mice increased with increasing light intensity (Fig. 7h), confirming the clear light response restoration of blind mouse retinas in vivo. The VEP amplitude in NW arrays-implanted blind mice was larger than that in blind mice, but still smaller than that in wild-type mice (Fig. 7i). We also observed light evoked spiking activities in the superior collicus (SC) in NW arrays-implanted blind mice (Supplementary Fig. 11).

Fig. 7 In vivo light responses after subretinal implant of NW arrays. a Explanted eye showing the position of the implanted NW arrays. b Slice of eyeballs in blind mice 7 weeks after implant surgery. c Dissected NW arrays-implanted eyecup 5 months after NW arrays implant surgery. d Brn3a and ChAT staining in higher magnification images showing the lamination of the NW arrays-implanted blind retina 5 months after implant. e Schematics of in vivo electrophysiology recording. The NW array was implanted subretinally, and multi-electrode array was inserted into the primary visual cortex to record light responses. f Raster plots and post stimulus time histograms (PSTHs) of spikes from V1 neurons in blind mice (left), NW arrays-implanted blind mice 2 days after implant (middle left), 2 months after implant (middle), 5 months after implant (middle right), and wild-type mice (right). The purple shade area indicates the presentation of light. g 15 traces of near UV light-evoked LFP responses in V1. Purple line indicates the presentation of light. h Percentage of stimuli that elicited responses in blind mice and NW arrays-implanted blind mice using different light intensities (Blind + NW: 637 μW mm–2, n = 6; 318.5 μW mm–2, n = 5; 159 μW mm–2, n = 5; 79.6 μW mm–2, n = 5. Blind: 637 μW mm–2, n = 6; 318.5 μW mm–2, n = 4; 159 μW mm–2, n = 4; 79.6 μW mm–2, n = 4. Two-tailed Student’s t-test, P 637 = 0.016). i VEP amplitude in NW arrays-implanted blind mice, blind mice, and wild-type mice (n = 5 for NW arrays-implanted blind mice, n = 5 for blind mice, n = 4 for wild-type mice. Two-sided Wilcoxon rank-sum test, P blind vs. blind+NW = 0.056). Data are presented as mean and S.E.M. Scale bars: 500 μm (a–c), 20 μm (d) Full size image

The vision in near UV was investigated in vivo in alive rd1/cDTA mice. Although cornea, lens and other tissues in the eye absorb some UV light, mouse retinas can still be sensitive to UV light36. The absorption of UV light (375/28 nm) in the cornea and lens of the mouse was estimated to be ~42%. The near UV light responses in V1 in anaesthetized wild-type mice were measured in vivo (Supplementary Fig. 12a). It is clear that near UV light triggered both LFP and spiking activities across V1 (Supplementary Fig. 12b, c), indicating that wild-type mice have vision in UV37.

Recovery of the light sensitivity in awake-behaving mice

In blind mice, the light-induced pupil constriction was largely impaired due to the loss of photoreceptors in the retina (Fig. 8a). Many retinal prosthesis studies use PLR to confirm the behavioral recovery of light sensitivity34,38,39,40. We implanted NW arrays subretinally into one eye of blind mice (Supplementary Fig. 13). The PLR to near UV light in the implanted eye was improved compared to that in the contralateral (control) eye at both 0.13 μW mm–2 and 0.25 µW mm–2 4–8 weeks after implant surgery (Fig. 8b, Supplementary Movie 2 in the Supporting Information). Likewise, the PLR to green light in the implanted eye was also improved at both 0.13 μW mm–2 and 0.38 μW mm–2 (Fig. 8c, d, Supplementary Movie 3 in the Supporting Information). Some of the implanted eyes reached the same level of PLR as that in wild-type mice, indicating the recovery of light sensitivity in multiple colors.