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1 1 Electronic Supplementary Material to the publication of E. Zrenner et al.: Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc.R.Soc.B (2010) at URL: Details on the Technology of the Subretinal Implant, Clinical Study Design, Results and Spontaneous Reports of Patients including nine Movie Clips on performance Eberhart Zrenner 1*, Karl Ulrich Bartz-Schmidt 1, Heval Benav 1, Dorothea Besch 1, Anna Bruckmann 1, Soeren Danz 2, Veit-Peter Gabel 3, Florian Gekeler 1, Heinz-Gerd Graf 4, Udo Greppmaier 5, Alex Harscher 5, Gernot Hoertdoerfer 6, Steffen Kibbel 5, Uwe Klose 2, Andreas Kopp 2, Akos Kusnyerik 7,1, Wilfried Nisch 8, Tobias Peters 9, Daniel Rathbun 1, Siegmar Reinert 10, Katarina Stingl 1, Helmut Sachs 11, Ieva Sliesoraityte 1, Alfred Stett 8, Peter Szurman 1, Barbara Wilhelm 9, Robert Wilke 1, Walter Wrobel 5 1 Centre for Ophthalmology, University of Tübingen, Schleichstr. 12, Tübingen, Germany 2 Dept. of Radiology, University of Tuebingen, Hoppe-Seyler-Str. 3, Tuebingen, Germany 3 Eye Clinic, University of Regensburg, Regensburg, Franz-Josef-Strauss-Allee 11, Germany 4 Institute for Microelectronics Stuttgart (IMS CHIPS), Allmandring 30a, Stuttgart 5 Retina Implant AG, Gerhard-Kindler-Str. 8, Reutlingen, Germany 6 MobilityTraining, Mozartweg 11, Tuebingen, Germany 7 Department of Ophthalmology, Semmelweis University, Tomo u ,1083-Budapest, Hungary 8 NMI Natural and Medical Sciences Institute at the University of Tübingen, Markwiesenstr. 55, Reutlingen, Germany 9 Steinbeis Transfer Centre Eyetrial at the Centre for Ophthalmology, Schleichstr , Tübingen, Germany 10 Department of Oral and Maxillofacial Surgery, University of Tübingen, Osianderstr. 2-8, Tübingen, Germany 11 Klinikum Friedrichstadt, Friedrichstr. 41, Dresden, Germany *Author for correspondence and requests for materials This document gives details on technical, clinical and outcome parameters concerning the study on subretinal implants in blind patients published in Proceedings of the Royal Society B by Zrenner et al. in 2010 (doi: /rspb ). A light-sensitive, externally powered microchip was surgically implanted subretinally near the macular region of volunteers blind from hereditary retinal dystrophy. The results of this study demonstrated for the first time that subretinal micro-electrode arrays with 1,500 photodiodes can create detailed meaningful visual perception in previously blind individuals. The additional information explains in further detail the active subretinal chip, the study s design, the results, information on the study s background and its supporters as well as the content of 9 web-based movie clips. Keywords: Subretinal neuro-prosthetics; artificial vision; bionic vision; retinal implant; retinitis pigmentosa; role of microsaccades; pupillography INTRODUCTION The subretinal implant used in the study by Zrenner et al. [1] contains an array of 1,500 active microphotodiodes ( chip ), each with its own amplifier and local stimulation electrode. At the implant s tip, another array of 16 wire-connected electrodes allows light-independent direct stimulation (DS) and testing of the neuron-electrode interface. Visual scenes are projected naturally through the eye s lens onto the chip under the transparent retina. The supplementary material provided here describes details on technical, clinical and outcome parameters concerning this study. Figures refer to the main publication [1], except those marked S.

2 2 1. TECHNICAL DETAILS CONCERNING THE SUBRETINAL IMPLANT (a) The 4 by 4 array for direct electric stimulation (DS): electrode dimension and stimulation characteristics As shown in figure 1 of the main publication [1] the distance from one quadruple DS electrode center to the next (280 μm) closely corresponded to 1 of visual angle [2] (figure 1c), so that the entire DS test field covered a visual field of 3.5 x 3.5, or roughly the retinal image size of a square approximately 3.6 cm x 3.6 cm viewed at a distance of 60 cm. The signal delivered by each electrode is controlled via a wireless receiver in the power supply box (see below). The reference electrode is located distally under the skin at the orbital rim (figure. 1b). Charge recovery is provided by grounding each electrode after each pulse, which drives a return current. Thus, monophasic voltage pulses (figure S1A a, top) effectively yield biphasic currents (figure S1A a, centre) and a charge delivery pattern shown in figure S1A a (bottom), driving the stimulating current from the distal to the proximal retina, polarizing bipolar cells that in turn excite ganglion cells as shown schematically in figure 2c of the main publication. This mode of selective stimulation of bipolar cells has been demonstrated previously through synaptic blockade [3]. Charge was calculated as the area under the current waveform (figure S1A a, center), driven by a constant voltage pulse, measured across a 1 kω resistor in the lead of the common return electrode. A typical maximum charge transfer per single electrode is in the range of 2 nc (in saline solution) and a charge density of 80 μc/cm² at the surface of the TiN electrode. (b) The light sensitive MPDA array: functional characteristics and output control Each of the 1500 elements ( pixel, see figure 1 of the main publication) generates monophasic anodic voltage pulses at its electrode which are linearly amplified up to 2.3 V, thus providing a stimulation current proportional to the intensity of light falling on each element. Activity of the chip is controlled by pulses from an external power supply provided via the cable and plug shown on the right of figure 1b. The conversion from light to voltage takes place for a period of 0.5 to 6 ms ( image recording time ) at a rate of 2 to 20 Hz (both externally adjustable). Consequently the entire image projected on the chip is sampled at that frequency with a spatial resolution of approximately 1,500 pixels. Thus, pixelized repetitive stimulation with a current injection pattern shown in figure S1a is delivered simultaneously by all electrodes to adjacent groups of bipolar cells [3], the amount of current provided by each electrode being poportional to the strength of light falling on the respective photodiode. Light levels ranging across approximately 2 log units are converted to charge pulses by each pixel with a sigmoidal relationship (figure S1A b and c; in vitro measurements). The operating range (i.e. sensitivity) can be shifted manually by several log units by applying an external control voltage Vglobal (VGL) to allow perceptual resolution of as many contrast steps as possible in various ranges of ambient illumination from 2 to 10,000 lux (figure S1A c). Additionally, a second control voltage (Vbias) adjusts the maximum output of the electrodes (amplifier gain) between approximately 1.25 and 3.5 nc in this example (figure S1A b). Sensitivity and gain are adjusted externally for all elements simultaneously. In addition, the input/output characteristics of the MPDA was investigated by means of electroretinography (ERG) in patients. Figure S1A. Properties of electric stimulation (a) Monophasic anodic voltage stimulus (top) and the resulting biphasic time course of current injection (center) as well as respective charge transfer (bottom). (b) maximum output at a given sensitivity can be adjusted from outside by varying gain control line VBias in between 1.4 and 2.01 Volt. (c) Sigmoidal dependency of charge created per pixel from illuminance in lux; the position of the function and thereby threshold sensitivity can be adjusted from outside by varying sensitivity control line VGL in between 1.82 and 0.61 V. Threshold voltage to elicit a percept was assessed in an up-and-down staircase procedure from 0.1 V up to 2 V (maximally 2.3 V in some cases) for each of the 16 electrodes. The lowest thresholds were found for biphasic, equal duty pulses (usually cathodic first) with a phase duration of 2 to 3 ms. Voltage threshold values for monophasic pulses were usually higher than biphasic pulses. Typical in vivo impedances of the 100x100 μm 2 DS electrodes were within a range of 30 to 300 kω at 1 khz.

3 3 a b 8 R-I (80/45) R-II (80/45) R-I (75/45) R-II (75/45) R-I (70/47) R-II (70/47) ERG Amplitude, nv Time, ms ERG Amplitude, mv Chip: 3050_17 F 3 b SNR: RI00126 f = 10 Hz PD = 0,4 ms Vbias = var. Vgl = var. 0,1 0, Luminance, cd/m 2 Figure S1B. (a) Typical potential change evoked by Ganzfeld light stimulation (1cd/m²) of the MPDA using electrophysiological (ERG) recording in-vivo by means of corneal electrodes (relative values on x-axis). The potential reflecting the electrical response of the MPDA is used for testing function of the device after implantation. The MPDA-signal is filtered ( Hz), in order to avoid patient related artifacts (b) Amplitudes shown in part a, plotted against luminance of the Ganzfeld stimulus. Full and dashed lines respectively represent two recordings (RI and RII) of MPDA signals with matching VBias and Vgl settings (shown in brackets on top) in order to test reproducibility. The subretinal MPDA was activated for 0.4 ms with a 10 Hz repetition rate to capture the light and to eject charges the dimension of which is related to stimulus luminance. In order to optimize sensitivity and gain settings, the Espion E² console and the ColorDome full field stimulator were used (Diagnosys Ltd UK, Cambridge, UK). Amplitudes of MPDAevoked potentials (figure S1Ba) were measured in vivo with increasing luminance of Ganzfeld test flashes varying from 0.1 photopic cd/m² to 1000 photopic cd/m² in log 0.5 steps. Highly reproducible VBias and Vgl settings were identified (figure S1Bb) that matched gain and sensitivity of the MPDA to the luminance distribution of the objects the patient was looking at. The shape of the in-vivo input/output functions were comparable to in-vitro measurements provided by the manufacturer (Retina Implant AG, Reutlingen, Germany), examples of which are shown in figure S1A. In relation to human perception, the spectral sensitivity of the photodiodes is almost flat in the visible spectrum, but slightly elevated in the near infrared. Thus, the chip is very sensitive to red and infrared light, making it possible to operate the chip with red or infrared light which is within the range of luminance used here- invisible to any original photoreceptors that might remain. (c) The power supply Upon command, the stimulation box shown in figure 2a and 2d of the main publication can release preprogrammed pulses to the DS test field or run the chip with a pre-defined set of parameters. It is controlled by the experimenter through custom software installed on a PC. The software allows the experimenter to set temporal parameters (polarity, duration and intervals) for both single pulses and sequences of pulses of the DS test field. A second software package allows the experimenter to turn the chip on and off and to set the desired parameters for the chip sensitivity and gain. All commands and parameters are logged. For recordings using the DS test field of electrodes, a measuring box was inserted between the stimulation box and the implant. The stimulation pulses could then be monitored and the current flow measured using a 1 kohm resistor in the common return electrode. 2. CLINICAL STUDY, CLINICAL PATIENT DATA, SURGICAL CONSIDERATIONS AND TESTING PROCEDURES (a) Clinical study design and approval This pilot study was approved by Tübingen University's Ethics Committee for a group of maximally 14 patients (registered at Safety assessment and function tests were conducted over four consecutive months. Presented here are data from the last three patients of this first pilot study; they were selected for this report because they received the latestgeneration implant with improved cable characteristics and an optimized chip seal. Study duration was 126 days including implantation and explantation surgery. For preoperative testing procedures, see below. Inclusion criteria were: Hereditary retinal degeneration of the outer retinal layers with the retinal vessels retaining perfusion and pigments of mild to moderate density, age years, blindness (at least monocular) or visual functions insufficient for navigation/orientation, period of appropriate visual functions > 12 years; Visual acuity 20/400 earlier in life, willing and able to give written informed consent and able to perform the study during the full period of 126 days. Exclusion criteria were: any other ophthalmological diseases with relevant effects upon visual function, systemic diseases that might imply considerable risks with regard to the surgical interventions and anesthesia, neurological and/or psychiatric diseases, known hypersensitivity to any of the ingredients of the study device, pregnant or nursing women or women of childbearing potential who were not willing to use a medically acceptable means of birth control for the duration of the study.

4 4 The study protocol comprised 33 trial visits with safety and efficacy assessments. Criteria used to evaluate safety were implant-related adverse events, tolerability of the implant and laboratory results from internal medicine. Criteria used to evaluate efficacy were light perception, temporal resolution, localization, motion perception and object recognition as assessed by specially designed tests for quantitative measurement in the low vision range (see Psychophysical Tests, below). (b) Preoperative procedures General function of the patients visual system had been tested pre-operatively by corneal electrical stimulation in order to elicit electrical phosphenes, following a previously described protocol [4]. In short, corneal DTL electrodes as commonly used in clinical ERG registering were placed on the inferior corneal limbus. A neuro-stimulator (Twister; Dr. Langer GmbH, Waldkirch, Germany) was used to deliver monopolar, positivefirst, rectangular current pulses at different pulse durations to subjectively assess overall retinal sensitivity to electrical stimulation. Values of currents necessary to elicit visual threshold sensations for different stimulus durations were then compared to our published values [4]. The presence of inner retinal cells, important for connection of subretinal electrodes to neurons, can be assessed by thickness measurements of the inner retina as well as by fluorescence angiography; if thin retinal arterioles and venoles can be seen and if OCT indicates paramacular retinal thickness of ~200 micrometer, presence and excitability of inner retinal neurons are very likely. All patients underwent cataract surgery and implantation of a posterior chamber lens in preparation for the study prior to implantation in order to obtain the best possible optic media and to prevent a secondary cataract due to the silicone oil introduced into the vitreous in the course of the study. (c) Patients clinical data Patients gave informed consent to participate in the study according to the Declaration of Helsinki. Patient 1 (40 yrs old, female) had typical bone spicule pigment formation in the retinal periphery and RPE atrophy. Examination through OCT revealed central retinal atrophy, but no macular edema. She had a suppressable intermittent nystagmus. Motility was free. Intraocular pressure was within normal limits (13/14mmHg). Phosphenes could be elicited electrically in only the right eye (50ms, 1.4mA). From the beginning of her 7th school year, she had problems with reading; she was able to drive a bicycle until the age of 15. At the time of implantation she navigated freely with the help of her guide dog. Objective refraction after chip implantation and oil endotamponade was +7.0sph -1.25cyl 5 in the right eye. Patient 2 (44 yrs old, male) was night blind at the age of 16. Military service at the age of 19 was performed with great difficulties at night. He got his driver s license at the age of 18 but didn t drive at night. At the age of 30 he had increasing difficulty recognizing faces and reading. This worsened to a lack of reading ability at the age of 38. At the time of surgery, he navigated with the help of a white cane and could locate light. Preoperative examination showed no secure light projection in both eyes. Anterior segments were normal apart from posterior cataract. Funduscopy revealed retinits pigmentosa with central atrophy and moderate bone spicules in the mid-periphery. The OCT showed central retinal atrophy and did not reveal macular edema. Intraocular pressure was within normal limits (12/12mmHg). Pupil reaction was noticeable in both eyes, with no RAPD. Motility was free. Phosphenes could be elicited electrically in both eyes. (O.D. : 25 ms, 1.2 ma ; 50 ms, 0.7 ma ; O.S. : 25 ms, 0.6 ma ; 50 ms, 0.55 ma). Objective refraction after chip implantation and oil endotamponade was +7.0sph -3.0cyl 120 in the right eye. Patient 3 (38 yrs old, male) had light perception in both eyes but no light localisation. He had not undergone previous surgery before the cataract extraction. Anterior segments were normal apart from posterior pole cataract. Funduscopy revealed choroideremia with widespread pigment atrophy, but a remaining central pigmentary island. The OCT showed central retinal atrophy and no edema. The pupils reacted slowly on light perception; there was no RAPD. The eyes were exotropic, and motility was free. Intraocular pressure was within normal limits (13/15mmHg). Phosphenes were elicitable electrically in both eyes (O.D.: 10 ms, 1 ma ; 25 ms, 0.6 ma ; 50 ms, 0.5 ma ; O.S. : 10 ms, 0.8 ma ; 25 ms, 0.6 ma ; 50 ms, 0.45 ma). The patient was healthy and did not use eye drops or systemic medication. He smoked cigarettes. He had noticed problems with night vision already at age of 6 years. He was able to drive a motorcycle until the age of 16 and a bicycle until the age of 18. He could read books without visual aids until the age of approximately 16 years. At the time of implantation he moved with the help of a white cane, used an audio and tactile response laptop and had learned to read Braille. (d) Preoperative optimization of chip localization It is very important to position the subretinal implant close to the macula at areas of presumably preserved inner retinal function, as outlined below. This requires individualized calculation of the intraocular length of polyimide foil. The procedure developed by Kusnyerik et al. [5] utilizes colour fundus photography, fluorescein angiography and 3D-OCT overlaid in a concordant grid map to analyze the retina. For every field of the grid, an averaged score is calculated and combined with expert assessment of retinal thickness, density of small retinal vessel and RPE-pathology - weighting each imaging modality by a factor. Imaging techniques were used to assess individual geometrical dimensions of the eyeball (ultrasound, interferometry, and high-resolution, 3 Tesla MRI). To create a 3D model of the eyeball, we applied the geometrical measurements to an ellipsoid eye model. This model was used to calculate the angle and distance along the surface of the ellipsoid between the scleral point of insertion and the desired location of the MPDA at the posterior pole.

5 5 The described preoperative planning led to successful placement of the MPDA at the retinal location of presumed greatest therapeutic potential. (e) General surgical considerations The curvature of the posterior part of the eye apparently presents no problem for rigid, planar devices of 3x3 mm² as seen in OCT images. The connection from the eye to a retroauricular power supply is unusual in ophthalmological surgery and takes extra time; although this procedure is neither new nor special as it is the state of the art in cochlea implant surgery. A one-piece, fully intraorbital device may be more attractive, as no interdisciplinary surgical approach is necessary. On the other hand, transmitting wireless energy to a moving eyeball, although feasible, is a formidable challenge of its own. The challenges include the limited space in the orbit for electronic components, the concentration of heat sources, and the surgical difficulty of delicately introducing the device into the subretinal space with a bulky piece of hardware connected just 3 cm away from the tip of the very flexible subretinal foil. At this time it seems preferable to have more space and freedom of movement by having the power supply and control signal line positioned behind the ear. (f) Postoperative observations and safety Safety was closely monitored during the study by daily funduscopy (figure 2f of the main publication), angiography at selected study visits and high resolution optical coherence tomography (OCT). Additionally, CT was used in some patients to reconstruct the cable position at 9 directions of gaze (example in figure 2e). There were no serious intraocular side effects of the implant, such as preretinal bleeding, persistent increase of intraocular pressure, intraocular inflammation, retinal detachment, or retinal neovascularisation in these patients. In patient 3, during implantation, a small circumscribed area of subretinal bleeding was observed at the posterior pole, with complete resorption during the following 10 days. In the same patient a mild skin infection of the retroaurical cable exit was reported after explantation of the chip, with restitution ad integrum after a few days. (g) MPDA: Psychophysical testing procedures The tests described in the main publication were performed separately in two conditions: with "Power ON" and "Power OFF" ( baseline performance ): 1. BaLM ( Basic Light and Motion Test ) was used to assess 4 basic visual abilities: a) light detection, b) basic temporal resolution (two flashes of light), c) object localization, d) movement detection. 2. Bright white stripes were presented in 2 directions (horizontal or vertical) or 4 directions (two additional oblique positions) for grating detection on a dark background in the BAGA-test (see figure 3a). Since the patients perceived thin white stripes with broader black areas between them more easily, the width ratio was adjusted between 1:2 and 1:5 (white:black) for each patient, individually. 3. Optotypes. The standardized FrACT (Freiburg Visual Acuity and Contrast Test) [6] uses Landolt C optotypes and an up-and-down staircase procedure to estimate the visual acuity in terms of maximum likelihood after 36 single trials. If the Landolt test was passed successfully, single letters were used subsequently. 4. Objects common in daily life: Patients were asked to recognize and localize the arrangement of items, i.e. geometric forms of equal area, dishes and flatware or high contrast fruit at different positions. The aim of this test was to evaluate the patient s ability to recognize objects common in daily life. Performance was rated on a scale by an independent, experienced mobility trainer. 5. Optional tasks were designed individually to explore each patient's ability to name shapes of a given size. After determination of the optimum size, a forced-choice test with 2-6 alternatives was used to determine with statistical significance the ability to discern shapes such as geometric objects, alphabetical letters, words made from such letters, hands of a clock or objects of different brightness. All objects were cut out of white (or grey) paper and presented on a black background. The patients were shown various white objects on a black nonreflective table (Setup 2, figure 4a, surface luminance 0.4 cd/m 2 ) at reading distance (45-50 cm). Searching head and eye movements were unrestricted in order to come as close as possible to realistic conditions. Chip settings were unchanged from task 3 and refraction was adjusted for reading distance. The patients were then interviewed and their performance assessed by an independent mobility trainer in a rigid protocol. In this task, patients were asked to recognize, identify, and arrange three types of highly contrasting objects: geometric forms, dishes and flatware, or fruit. This experimental paradigm also included eye-hand coordination tasks and haptic feedback. While the patients were first asked to describe the scene without touching any objects, they were then encouraged to grasp objects and verify and correct their description. Subsequently the order of objects was rearranged and the test repeated. Although significant learning effects in hand-eye coordination could be observed, those effects could not yet be assessed in a statistical manner in this first pilot trial, and were instead assessed by an independent professional mobility trainer, who over time clearly noticed improvements of various degrees in these patients in daily life tasks. Apparently there are different types of learning effects, some of which occur within days while others may need weeks and months. Less demanding tests were completed first in order to allow each subject to adapt. Due to this hierarchical structure of testing, results are not available for all tests in all patients. Since some patients had residual perception of bright white light in the periphery of the visual field, red light with a cutoff at 635

6 6 nm(schott filter OG 635) was used for making sure that remaining, natural photoreceptors were not recruited in tasks using screen projection (tasks 1, 2, 4). As the sensitivity of natural photoreceptors is very low compared to the chip at this wavelength (~1000-fold difference), these control experiments, where in the chip-off condition there is no sensation whatsoever, preclude any possible contribution of residual photoreceptor function. Additionally, neutral density filters (Schott NG filters log U) served for brightness attenuation. Maximum screen luminance was ~3,200 cd/m 2 (for white light). Chip settings were adjusted for a working range of 8 to 800 cd/m 2 white light or 1.2 to 4.3 cd/m 2 red light, with objects projected on a screen at a viewing distance of approximately 60 cm. Since the photodiodes are also sensitive to long range wavelengths of the visible spectrum, red light was quite effective to allow perception in persons with subretinal implants while appearing dim to a normal observer (see method section). Luminance was set individually in such a way that it did not elicit a visual sensation in patients at all when the chip power was switched off. Because only a single spectral type of photosensor is built into the chip, both white and red light elicited very similar percepts. (h) Validity of psychophysical testing procedures Use of a schematized testing procedure, as realized by the psychophysically elaborated BaLM, BAGA and FrACT tests with multiple alternative forced testing procedures has turned out to be very useful in providing statistically sound, reliable and reproducible data. With some improvements concerning flexibility of stimulus presentations such as relation between dark and light areas, duration of presentation and intervals, this type of testing may prove to be very helpful to compare the various prosthesis approaches. In particular, comparing test results with devices in a powered and unpowered state, the fact that the respective states are not known to the investigating person is an important tool to improve reliability. In BaLM, BAGA and FrACT tests this is not a problem anyhow, as the test automatically presents a series of optotypes of different sizes in an up and down procedure independent of the investigator. We also have learned that a hierarchical order according to the difficulty of the test is important, divided into mandatory and optional tasks. It makes no sense, given the finite concentration and attention resources of a patient, to schematically run through all tests from beginning to end. Therefore we have ordered the tests according to difficulty. A patient who cannot localize a plate or a cup has not been asked to read letters. This does not mean that tests have been done erratically but that each patient was guided only to the level of his/her maximal performance in a logical and sequential manner. Although it may be criticised that not all data are available in all patients, all data available (except where indicated) are properly controlled by 2 or 4 AFC. However, it would have been asking too much from our patients to subject them to hours and days of tasks that they clearly cannot perform (e.g. the 15 minute presentation of Figure S2. Pupillary light reflex amplitude (mean and SD) of patients 1-3 evoked by stimulation at corneal light levels of 84 or 380 lux. Each patient was tested with optimum stimulus intensity for the individual MPDA characteristics. Patients 2 and 3 showed residual pupillary light reactions even when the chip power was switched off. 36 objects in an up and down 4AFC procedure when the patient exhibits absolutely no recognition for a certain task). It can be expected that more and more patients may be included in electronic implant studies that have some remnant light sensitivity, allowing them to differentiate dark from bright surroundings without being able to recognize large objects. In such cases it may be advisable to use red light for testing in certain situations. Red stimuli can easily be adjusted in brightness so that they are not detected by rods or cones that are very insensitive in the long wavelength end of the visible light spectrum while the implant employs microphotodiodes that have high sensitivity in this spectral region. (i) Pupillography Infrared videopupillography (CIP, AMTech, Germany) was used to quantify light-induced pupillary responses due to MPDA activation at corneal illuminations of 18, 84, and 380 lux of 200ms duration. After an adaptation period of five minutes to a background light of 2 cd/m 2, four pupil responses to light were recorded and averaged for each stimulus intensity. After each stimulation, the patient also reported on his/her visual perception. The results are shown in figure S2. All three patients showed stronger pupillary constrictions at the chip-on condition and they reported simultaneous light perception. (j) Nystagmus Unintended eye movements (nystagmus) are commonly observed in blind persons. The problem of patient 1 to recognize grids may be at least partly explained by her unsteady fixation and because there is no fixation target for the eye to rest upon. We observed that such chronic, unintended eye movements tended to cease during the testing period when visual objects could once again be used for fixation. However, if such eye

7 7 movements are initially high in amplitude and frequency a fixation-learning process may be impeded to some extent. Projecting bright lines directly onto the chip under visible fundus control enabled patient 1 to compensate for some of the eye movements; and in that way bypassed the requirement of stable fixation, allowing Pat 1 to recognize line direction correctly with the MPDA. 3. SUMMARY TABLE OF THE RESULTS AND SPONTANEOUS REPORTS OF PATIENTS (a) Summary table Table ST1 provides a summary of the results described in the Result section of the main publication [1]. Spatial discrimination is best within the macula due to its cortical overrepresentation. The implant was located closest to the macula in Patient 2, who also had the best spatial resolution of the 3 patients. Distortion due to the particular arrangement of bipolar cells in the foveola is apparently not a major problem, as bipolar cells are less displaced from photoreceptor localization than ganglion cells. Moreover there are only a few electrodes covering the foveola because the distance between electrodes is 70 micrometers. During the grating test (figure 3b) Pat. 2 reported only two, sometimes three parallel lines due to his limited visual field. However, he described them as appearing immediately without delay or the need for eye or head scanning, and clearly visible as a grid without distortion (see Ch.5, Movie 3); apparently putting a chip with 70 micrometer electrode spacing under the retina does not create a physiological problem. (b) Statistical evaluation (d) Spontaneous reports of patients and learning effects Whenever forced choice tests were performed, the number of alternatives (choices) is indicated. Based on the number of choices and the number of repetitions of a test, a respective p- value is calculated with the binomial function, indicating the likelihood that the respective hit rate was achieved purely by guessing. Furthermore, for all results of forced choice tests, the chance rate as well as the hit rate to reach the psychometric threshold are indicated. Psychometric thresholds were calculated as (chance rate + ½ (100%-chance rate)); this defines the point above the rate of mere guessing at which 50% of remaining tasks were solved correctly [7]. (c) Contrast and spatial resolution It must be noted that patients succeed much better at recognizing white objects on a black background than vice versa. This may be caused by the superposition of electric fields between simultaneously stimulated, neighbouring electrodes when black lines are presented on a white background, thus decreasing electrical stimulus contrast. With white lines on a black background, each line has a non-stimulated area next to the line so that the contrast is much better. So far in all retinal implants each electrode stimulates a group of neurons containing both ON and OFF cells that normally respond with opposite polarity and therefore may cancel out each other s responses. Nevertheless patients were able to see brightness contrasts quite well, probably due to the following facts: first, the population of OFF-bipolar cells is smaller than that of the ON-bipolar cells which dominates the response; second, a single pathway is able to mediate contrast vision, as observed in patients with congenital stationary night blindness who have only functioning OFF cone bipolar cells [8], third, stimulation only lasts milliseconds, similar to very brief repetitive ON/OFF flashes e.g. in a dancing bar, which excite ON and OFF bipolar cells quasi-simultaneously, and can nevertheless transmit the perception of a scene in an otherwise dark room. Contrast could be improved by bipolar electrode configurations that provide more local restriction of current flow instead of the monopolar electrode array used in this study, and by asynchronous activation of individual pixels. Although several observations by the patients and investigators could not be quantified in this first pilot study, some of them may be worth reporting beyond those presented in the main manuscript. All patients recognized some flickering with MPDA vision that resulted in a slightly fluttering image which they reported acclimating to and which was constantly there without special eye or head movement. Moreover they learned quickly where to expect a target in the visual field and turn their attention to this particular area, controlling their gaze direction. Patient 1 was unable to discern objects presented on a screen for quite a long time and recognized shapes only under controlled conditions (i.e. when objects were directly projected onto the chip). However, it is possible that her slight nystagmus and an inability to perform adequate head and eye movements impeded stable capture of the scene with the small visual field. Pat. 2 reported that letters look like a wobbly image similar to a situation looking at large targets on the floor of a swimming pool through small breaking and moving waves on the water surface; letters would not lose their general shape but would show constant slight movements within their various parts. He also reported that he could read slightly smaller letters better than large letters, as they fit more easily into his restricted visual field as a complete object, but this may render it more difficult for some letters to be discerned from others. He learned to take advantage of his newly regained abilities to a degree that enabled him to localise and name objects and letters within a few seconds, but sometimes it took up to 2 minutes to find and double-check letters or words. However, he usually felt that double-checking did not change the answer. In one remarkable event he spontaneously gazed at his hand and described his fingers as multiple straight lines following the movement of his hand in all directions as well as to a side view, bending his fingers slowly, reporting the fascination of seeing his hand again after so many years (see Ch. 5, Movie 9). When freely moving around in a room, he was able to discern a larger person in a white coat from a smaller one and to see which arm was lifted if this person moved his/her arm upwards.

8 8 Direct Electrical Stimulation via the DS-array (4 by 4 electrodes) Perception of single electrical pulse at single electrode Single pulse, row of 4 electrodes (horizontal or vertical) Single pulse oblique line Pat. 1 Pat. 2 Pat. 3 yellowish round dot yellowish round dot yellowish round dot / short line 2AFC 2AFC 2AFC correct angle of correct angle of correct angle of horizontal or vertical horizontal or vertical straight horizontal or vertical straight line line straight line correct orientation dark space between dots correct orientation dark space between dots correct orientation no dark space between dots Pattern, letter U in four directions correct Correct Failed (4AFC) Multiple letters partly seen Correct not able Sequential stimulation clockwise vs. not able Correct not able counterclockwise Light patterns projected onto the Microphotodiode Array Light detection BALM double flash (2AFC) CR: 50% PT: 75% ON: 81.3 %, passed OFF: 50 %, failed ON: 100 %, passed OFF: 37%, failed Localization of Quadrants in BALM Test (4AFC) CR: 25% PT: 62.5% BALM-movement test (4AFC) CR: 25% PT: 62.5% Grid direction detection(4afc) CR: 25% PT: 62.5% Landolt C ring staircase procedure single letters 8.5 cm high (4AFC) CR: 25% PT: 62.5% geometric objects on a table (4AFC) CR: 25% PT: 62.5% localization of dishes / flatware single letters cut out of paper presented on a table, 5-8 cm (16 AFC) CR: 6.3% PT: 56.3% Hands of a clock (12 AFC) CR: 8.3% PT: 58.3% 9 shades of grey (2AFC) CR: 50% PT: 75% as patient had problems with fixating small spot ON: passed for large grids 11/14 (2AFC) OFF: failed ON: 87.5%, passed OFF: ON: 63%, just passed OFF: 17 %, failed 0.34 cycl/deg: ON: 100%, passed OFF: 0%, failed 0.46 cycl/deg: ON: 62.5%, passed OFF: 12.5%, failed failed ON: logmar 1.69 OFF: failed, not measurable failed 3 AFC ON: passed Optional Tasks ON: 92 %, passed OFF: 29 %, failed ON: 100 %, passed OFF: 0 %, failed 4 AFC, ON: passed ON: 61 %, passed OFF: (5 AFC), 0%, failed ON: 92%, passed OFF: 8%, failed 7 shades differentiated ON: 78%, passed OFF: 40%, failed ON: 100 %, passed OFF: 62.5 %, failed ON: 25 %, failed OFF: 38 %, failed ON: 25 %, failed OFF: 50 %, failed 0.22cycl/deg: ON: 60%, almost passed, OFF: failed failed, but reported to have seen Landolt ring gap quite often clearly Failed 2 AFC ON: passed Table ST1. Summary of the visual functions observed in patients 1 to 3 in chip power ON and OFF conditions, respectively, as described in detail in the text. (AFC: alternative forced choice method; : not done; NA: not applicable; CR: chance rate; PT: psychometric threshold for seeing; log MAR: Minimum angle of resolution).

9 9 (e) Role of Microsaccades Besides high frequency tremor there are two important involuntary eye movements even during strict fixation [9]: Slow drifts with an amplitude of 2.5 min of arc and a velocity of 2 to 8 min of arc/sec as well as microsaccades with an amplitude of 3 to 50 min of arc and an amplitude-dependent velocity of 8 to 80 min of arc/sec. Such events occur 1 to 3 times per second. With the image refresh rate of 5-7 Hz found in our implant, patients report the perception of a slightly flickering, but constantly visible image - apparently continuously refreshed by such natural micro-saccades of the eye. The ability to engage normal eye movements for image stability in subretinal light sensitive implants avoids continuous head shaking necessary to refresh the image in spectacle-mounted camera systems, as discussed in detail in the main manuscript. 4. CONTRIBUTORS, FUNDING AND DISCLOSURE OF INTERESTS (a) Contributors For valuable contributions, we are very grateful to Mrs. Ursula Brunner, the members of the surgical team, the engineers and technicians, and first and foremost the patients; to Retina Implant AG in Reutlingen, Germany, who assembled and tested the implant, for sponsoring the study and for continuous support regarding technical function tests and experimental parameters, in particular to Dr. Walter Wrobel, Reinhard Rubow, Stefan Koberstein, Sonja Meine and Holger Wagner and Dr. Anuschirawan Hekmat, who monitored accordance with ICH 2003 regulations; to Prof. Michael Bach for developing the BaLM and BaGA software used for psychophysical testing; to Regina Hofer for art work; to Gernot Hörtdörfer, the independent mobility trainer who performed all daily life activity and orientation tests and helped to develop the test battery; to Heinz-Gerd Graf, Dr. Alexander Dollberg, Jan Dirk Schulze-Spüntrup, and Prof. Bernd Hoefflinger from IMS in Stuttgart, to Dr. Claus Burkhardt, Prof. Hugo Haemmerle, Dr. Gerhard Heusel, Dr. Wilfried Nisch and Dr. Martin Stelzle from NMI Reutlingen and Prof. Albrecht Rothermel of the University of Ulm for helping to design and fabricate the retinal implant and the prototypes; to Multi Channel Systems MCS GmbH in Reutlingen for contributing electronic components, to Dr. Thomas Zabel for support in the initial phase of the study, to Prof. Siegmar Reinert for helping to establish the extraocular surgical procedure and to Prof. Ulrike Ernemann, Dr. Soeren Danz, Dr. Andreas Kopp and Prof. Uwe Klose from the Dept. of Radiology at the University of Tübingen for imaging the eye and the chip, i.e. preoperative MRI data on dimension of the eye and reconstruction of the chip and the cable by 3D CT imaging (example provided by A.K. shown in figure 2). R.W. and E.Z. contributed equally to the paper. We are grateful to Professor Daniel N. Robinson, Oxford and to Dr. Daniel Rathbun, Tübingen for their invaluable comments. (b) Funding This work was supported by the German Federal Ministry of Education and Research (BMBF) in the form of grants Figure S3. Sketch of the typical relation of the letter X read by patient 2 to the size of the light sensitive MPDA during fixation. Physiological microsaccades and drifts, involuntarily occurring during fixation up to 50 min of arc cause the letter wobbling across the MPDA approximately 3 times per second. If the typical microsaccades and drifts according to Pritchard [9] are applied (inserted trace), the wobble amounts up to 4 pixels; this continuous change of retinal localization of the image, occurring in healthy volunteers as well, can prevent fading of the image occurring if images are stabilized on the retina. 01IN502A-D and 01KP to the Universities of Regensburg, Stuttgart, Tübingen and NMI, and grants 01KP0401, and to Retina Implant AG, and by the Kerstan Foundation, ProRetina Deutschland e.v., and the German Research Foundation (DFG) with a starting grant WI 3460/2-1 to R.W. (c) Disclosure of Interest Disclosure of possible financial interests: E.Z., V.P.G., F.G., H.G.G., W.N., H.S., A.S., W.W. hold minor amounts of stocks in Retina Implant AG. U.G., A.H. and W.W. are employed by Retina Implant AG; none of the other authors is paid for consulting or any other services to Retina Implant AG. E.Z., V.P.G., F.G., H.G.G., H.S., A.S., W.N. are inventors of initial patents acquired from the University of Tübingen by Retina Implant AG. Inventors of patents submitted later by Retina Implant AG: U.B.-S., P.S., R.W., A.H., W.W. The University Eye Hospital Tübingen, NMI, STZ, have received restricted grants by Retina Implant AG for performing preclinical experiments and the clinical pilot trial. 5. SHORT MOVIE CLIPS ON PATIENT PERFORMANCE Movie 1: Electrical stimulation via the 4 x 4 direct stimulation electrode array. Orientation of the letter "U". With the patients eyes covered, the electrodes of the DS-array were activated to form a "U" with its opening facing up, down, left or right. The

10 10 patient was asked to position a cardboard "U" with the same orientation. A 25- second period was allowed between presentations for the patient to make a decision and the bioengineer to set up the pattern for the next direct stimulation. Patient 1 is shown here; the remaining movies show Patient 2. (Video at Movie 2: Electrical stimulation via the 4 x 4 direct stimulation electrode array. Presentation of 5 different letters. The letters I,L,O,T, and V were presented singly to the patient by sequentially activating electrodes with single pulses, using fixed voltage increments above the previously determined threshold. Pulse durations were 4 to 7 ms, with 208 ms between the activation of two electrodes. The sequence was shown only once per run. After one letter, a period up to 45 s was provided for the patient to give his answer, and for the bioengineer on the left (not shown in the movie) to set up the next letter, indicated silently by the investigator. The letters combine to the word VOLT, correctly named by the patient but never presented previously to him. (Video at Movie 3: Recognition of grating direction. Gratings consisting of thin stripes were presented on a screen about 60 cm from the patient. The patient was asked to choose one of four possible directions presented in a random, preselected order by the investigator. Due to the limited visual field provided by the chip, the patient s field did not cover the entire screen. He usually saw only two lines at a time. (Video at Movie 4: Measurement of visual acuity with Landolt rings. The patient was asked to localize the gap in a Landolt "C" ring that appeared randomly in one of four different directions: top, bottom, left right. Visual acuity was calculated from the smallest Landolt C ring that was seen with 62.5% correct responses. (Video at Movie 5: Recognition of geometric structures. Geometric figures with an identical surface area (square, triangle, circle, rectangle, diamond) were shown to the patient, whose task was to identify each. The first two minutes of the searching process are not shown here. (Video at Movie 6: Objects from daily life. A table setting (saucer, spoon, knife, and cup) was presented to the patient on an illuminated black cloth. The relative positions of the objects were not told to the patient, whose task was to find and name them. (Video at Movie 7: Recognition and localization of unknown objects. Indiscriminately selected objects (cutlery, dishes, fruit, office supplies, etc.) were placed on a table in front of the patient, who was not told what they were. The patient was asked how many objects he/she perceived, to name and to locate them. Due to his small visual field he needed some time to first identify the number of objects and their location and to be sure that he had not overseen a possible third or fourth object. (Video at Movie 8: Letter recognition. A marker pen was used to write white letters on a black cardboard. The letters MIKA were written with a height of about 8 cm, and placed on the table in front of the patient. He recognized the name and identified the wrong spelling, as his name is MIIKKA, while the former Formula 1 driver Häkkinen, as mentioned by the patient, is written MIKA. Due to his small visual field he needed some time to first identify the number of letters and their location. For this task he needed about 2 minutes; the movie starts with the last minute of the searching process. (Video at Movie 9: Patient gazes at his hand. The patient during an experiment with unknown objects on the table suddenly discovers his hand and looks at it with visible joy, describing what he is seeing. (Video at REFERENCES 1. Zrenner, E., Bartz-Schmidt, K. U., Benav, H., Besch, D., Bruckmann, A., Gabel, V. P., Gekeler, F., Greppmaier, U., Harscher, A., Kibbel, S., et al Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc R Soc B. (doi: /rspb ) 2. Drasdo, N. & Fowler, C. W Non-linear projection of the retinal image in a wide-angle schematic eye. Br J Ophthalmol 58, Stett, A., Barth, W., Weiss, S., Haemmerle, H. & Zrenner, E Electrical multisite stimulation of the isolated chicken retina. Vision Res 40, (doi: /S (00) ) 4. Gekeler, F., Messias, A., Ottinger, M., Bartz-Schmidt, K. U. & Zrenner, E Phosphenes Electrically Evoked with DTL Electrodes: A Study in Patients with Retinitis Pigmentosa, Glaucoma, and Homonymous Visual Field Loss and Normal Subjects. Invest. Ophthalmol. Vis. Sci. 47, (doi: /iovs ) 5. Kusnyerik, A., Greppmaier, U., Klose, U., Bartz-Schmidt, K. U., Wilke, R., Sachs, H., Hekmat, A., Bruckmann, A., Gekeler, F. & Zrenner, E Preoperative 3D Planning of Implantation of a Subretinal Prosthesis Using MRI Data. Invest Ophthalmol Vis Sci 49:E-Abstract Schulze-Bonsel, K., Feltgen, N., Burau, H., Hansen, L. & Bach, M Visual acuities "hand motion" and "counting fingers" can be quantified with the freiburg visual acuity test. Invest Ophthalmol Vis Sci 47, (doi: /iovs ) 7. Green, D. M. & Swets, J. A Signal Detection Theory and Psychophysics. New York,NY: Wiley. 8. Zeitz, C., Kloeckener-Gruissem, B., Forster, U., Kohl, S., Magyar, I., Wissinger, B., Matyas, G., Borruat, F. X., Schorderet, D. F., Zrenner, E., et al Mutations in CABP4, the gene encoding the Ca2+-binding protein 4, cause autosomal recessive night blindness. Am J Hum Genet 79, (doi: /508067) 9. Pritchard, R. M Stabilized images on the retina. Sci. Am. 204, 72-78

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