When natural photoreception is disrupted, as in outer-retinal degenerative diseases, artificial stimulation of surviving nerve cells offers a potential strategy for bypassing compromised neural circuits. Recently, light-sensitive proteins that photosensitize quiescent neurons have generated unprecedented opportunities for optogenetic neuronal control, inspiring early development of optical retinal prostheses. Selectively exciting large neural populations are essential for eliciting meaningful perceptions in the brain. Here we provide the first demonstration of holographic photo-stimulation strategies for bionic vision restoration. In blind retinas, we demonstrate reliable holographically patterned optogenetic stimulation of retinal ganglion cells with millisecond temporal precision and cellular resolution. Holographic excitation strategies could enable flexible control over distributed neuronal circuits, potentially paving the way towards high-acuity vision restoration devices and additional medical and scientific neuro-photonics applications.

Computer-generated holography is an emerging technology for stimulation of neuronal populations with light patterns. A holographic photo-stimulation system may be designed as a powerful research tool or a compact neural interface medical device, such as an optical retinal prosthesis. We present here an overview of the main design issues including the choice of holographic device, field-of-view, resolution, physical size, generation of two- and three-dimensional patterns and their diffraction efficiency, choice of algorithms and computational effort. The performance and characteristics of a holographic pattern stimulation system with kHz frame rates are demonstrated using experimental recordings from isolated retinas.

Holographically patterned PAINTS could potentially provide a means for minimally intrusive control over neuronal dynamics with a high level of spatial and temporal selectivity.

Recent studies highlight the importance of the temporal domain in visual processing. Critical Flicker-Fusion Frequency (CFF), the frequency at which a flickering light is perceived as continuous, is widely used for evaluating visual temporal processing. However, substantial variability in the psychophysical paradigms, used for measuring CFF, leads to substantial variability in the reported results. Here, we report on a comprehensive comparison of CFF measurements through three different psychophysical paradigms: methods of limits; method of constant stimuli, and staircase method. Our results demonstrate that the CFF can be reliably measured with high repeatability by all three psychophysics methods. However, correlations (r = 0.92, p≪0.001) and agreement (Bland Altman test indicated 95% confidence limit variation of ±3.6 Hz), were highest between the staircase and the constant stimuli methods. The time required to complete the test was significantly longer for the constant stimuli method as compared to other methods (p < 0.001). Our results highlight the suitability of the adaptive paradigm for efficiently measuring temporal resolution in the visual system.

Neuroprosthetic retinal interfaces depend upon the ability to bypass the damaged photoreceptor layer and directly activate populations of retinal ganglion cells (RGCs). To date, the preferred approach to this task largely relies on electrode array implants. We are currently pursuing two alternative methods for light-based direct activation of the RGCs. The first method is based on applying caged glutamate over the retina and uncaging it locally to obtain RGC excitation. The second method is to artificially cause RGCs to express Channelrhodopsin II (ChR2), a light-gated cation channel. In addition to being non-contact, optical techniques lend themselves relatively easily to a variety of technologies for achieving patterned stimulation with high temporal and spatial resolution. Using the Texas Instruments Digital Light Processing (DLP - DMD) technology, we have developed an optical stimulation system capable of controlled, large-scale, flexible stimulation of the retinal tissue with high temporal accuracy. In preliminary studies, we are performing patterned photo-stimulation experiments using samples of caged fluorescent probes and in rat retinas that were virally transfected with ChR2.

Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications. The in vivo two-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue. However, to date, this has only been possible using relatively complex adaptive-optics systems. Here, the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality, optically sectioned fundus images. By remotely scanning the focus using an electronically tunable lens, high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired, thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging. Moreover, the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator, GCaMP6s. These results and the simplicity of the new add-on optics are an important step toward several structural, functional, and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.

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The new system could prove to be a basic tool for non-invasive in vivo small animal retinal imaging in a wide array of translational vision applications, including the tracking of fluorescently tagged cells and the expression of gene-therapy and optogenetic vectors.