This PDF file contains the front matter associated with SPIE Proceedings Volume 10866 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Recent advances in optoelectronic materials, device designs and assembly techniques allow for electronic/optoelectronic systems capable of establishing intimate, chronically stable interfaces to the brain. This talk summarizes recent progress in two areas (1) cellular-scale optoelectronic devices that inject into targeted regions of the deep brain for optogenetic stimulation/inhibition and wireless recording of neural activity and (2) thin, conformal sheets of electronics that laminate onto the surfaces of the brain for large-area, high-speed mapping of electrophysiological behavior.

For decades, genetically encoded Ca²⁺ indicators (GECIs), have been under development for the purpose of visualizing intracellular Ca²⁺ dynamics. Fluorescent GECIs are limited for the purpose of deep tissue long-term whole-body imaging due to their requirement for external illumination. Bioluminescent GECIs overcome these restraints but are somewhat compromised in terms of spatiotemporal resolution compared to fluorescent probes. To address this, we developed a bimodal Ca²⁺ indicator by combining a single fluorescent protein based Ca²⁺ indicator and a split luciferase. The novel design of this bimodal indicator enables Ca²⁺ imaging in the same specimen in both fluorescent and bioluminescent mode. The ability to switch between fluorescent and bioluminescent modes with a single indicator should benefit applications where micro and macro scale observation of cells or tissues is desirable. Use of such a probe enables trans-scale imaging, where macro scale imaging of a group of cells or tissue can be combined with fluorescent imaging of single cells.

Stable large-scale optogenetic interfaces for non-human primates (NHPs) have a great potential to answer fundamental questions about brain function and to develop novel therapies for neurological disorders. We have previously reported an interface that enables manipulation and recording from up to 2 cm² of cortical tissue by combining three technologies: 1- convection enhanced viral delivery to achieve high levels of expression across large cortical areas, 2- semi-transparent micro-electrocorticographic arrays to record from these expressing areas, and 3- artificial dura to protect the brain and provide optical access. Although this interface provided a unique platform to study network activity and brain connectivity, it was based on day-to-day implantation and explantation of the recording array which led to accelerated tissue growth on top of the brain and limited the efficient time window for optical access to only several weeks. We then needed to wait for a month or two to remove the tissue from the surface of the brain and regain optical access. Here, we are optimizing this interface by incorporating the recording array into the artificial dura to reduce the manipulation at the brain surface and increase the efficient optical access window to 3-9 months. We are using a transparent, flexible polymer as an insulator for our recording sites that can be easily molded into the artificial dura. Furthermore, we have optimized our stimulation setup to increase the number of simultaneous light stimulation locations. We believe this optimized interface has a great potential for long-term optogenetic experiments in non-human primates.

Conference Presentation for "Light sheet theta microscopy for quantitative imaging of large cleared samples"

Optogenetics allows for the study of signaling pathways during a variety of cellular processes. Here we use subcellular optogenetics to study the role of vesicular trafficking in cytokinesis. Optogenetically activating RhoA in the cell midline induces cytokinesis and intercellular bridge formation. RhoA activates the actomyosin contractility network, leading to retrograde plasma membrane flow, localized decrease in membrane tension, and increase in endocytosis at the cell middle. Endocytic vesicles and trafficking proteins localize to the intercellular bridge. Perturbation of these proteins inhibits furrow formation. Thus polarized endocytosis and exocytosis in the first steps of cytokinesis provide new membrane to the middle of the cell and allow the cell to build the cleavage furrow and intercellular bridge.

Optogenetic tools have been gaining popularity, in part because they can be used to decipher the wiring of signaling pathways. They are based on the ability of photoactivatable proteins to change their conformation and binding affinity when illuminated with light. Fusing these proteins to signaling elements allows for the specific regulation of a single player within complex intracellular signaling pathways. Consequently, a signaling pathway can be studied with high temporal and spatial resolution. Most cell-based optogenetic studies utilize microscopy-based methods combined with culturing in the presence of light, followed by biochemical analysis. In contrast, a flow cytometer singularizes cells along a capillary and measures cell size, granularity and fluorescence intensities. This method has major advantages over microscopy or biochemical methods, including the ability to analyze thousands of living cells at single cell resolution in a very short time. Hence, it is desirable to combine optogenetics with flow cytometry. To our knowledge, there is no established protocol for optogenetic flow cytometry. A broadly accepted procedure is to manually illuminate cells from outside the reaction tube with flashlight devices. However, manual illumination in the flow cytometer requires the light to pass through the reaction tube and, for live cell imaging, a cylindrical, heated water chamber. This causes substantial light scattering and loss of light. Moreover, the light intensity provided by manual illumination is not reproducible between experiments (angle, distance, etc.) and there is a practical limit to the number of wavelengths in one experiment. By constructing the pxONE prototype, we were able to overcome these limitations. With this device, cells can be illuminated with specific wavelengths in a temperature-controlled manner during flow cytometric measurements. This allows for precise and reproducible amounts of light within and between experiments. To demonstrate the utility of our device, we recorded the fluorescence signal of Dronpa in Ramos B cells during photoswitching. Ramos B cells are derived from a human Burkitt's lymphoma. Dronpa is a fluorescent protein that exists as a monomer, dimer or tetramer. In its monomeric form, it is non-fluorescent. Illumination with 400 nm light induces dimerization and tetramerization and renders the Dronpa protein fluorescent. This process can be reversed by illumination with 500 nm light. The Dronpa protein has been used to control the function and location of signaling proteins. We expressed a Dronpa-Linker-Dronpa protein in Ramos B cells to study photoswitching of Dronpa in a flow cytometer. Using our device, we were able to efficiently and reproducibly photoswitch Dronpa while recording its fluorescence intensity in real time. This method provides substantial advantages over current illumination protocols with manual illumination and significantly broadens the experimental repertoire for optogenetic tools and cage compounds. The first publication using our device was recently published and shows optogenetic regulation of T cells (https://www.biorxiv.org/content/early/2018/10/01/432740). Using our technology will significantly simplify and accelerate the discovery and development of novel optogenetic tools.

Pulsed infrared (IR) light in the 1.8 - 2 μm region can modulate neural activities with high spatial and temporal precision. However, the mechanisms underlying these photothermal interactions are not fully understood. Here we investigate the IR modulation of axon and muscle activities using the crayfish (Procambarus clarkii) opener neuromuscular preparation. A modulated fiber coupled laser diode (λ = 2 μm) is used to deliver pulsed light with durations between 10 – 500 ms. Twoelectrode current clamp (TECC) is performed to stimulate and monitor the neural activities. Laser-induced temperature changes are measured by an open patch pipette simultaneously with TECC. We find that IR pulses can reversibly inhibit or block axon (Na⁺ ) and muscle (Ca²⁺) spikes. In axons, single IR pulses can suppress the action potential (AP) amplitude and duration and increase the interspike interval. In addition, the rates of AP depolarization and repolarization are also modulated by IR pulses. Individual IR pulses can also block muscle fiber Ca²⁺ spikes. The IR-induced decrease in the input resistance (8.4%) can be a contributing factor for the inhibition phenomena reported here.

Infrared neural stimulation (INS) is a promising neuromodulation technique capable of exciting neural tissue without the need for exogeneous agents or genetic modification. Due to its high spatial specificity, INS could improve upon traditional methods of selective neural stimulation in both the laboratory and the clinic. As of yet, no study has compared the efficacy and safety of using different INS parameters such as spot size and wavelength. Moreover, differences in the methods of determining energy deposition and laser spot size make it difficult to compare stimulation parameters used in the current literature. Here, we present results comparing INS efficacy using 1450nm and 1875nm light over a range of spot sizes and radiant exposures. Stimulation thresholds were determined using recorded compound muscle action potentials (CMAPs) and visible muscle contractions in an in vivo rat sciatic nerve model. Overall, 1450nm light required lower radiant exposures to achieve threshold activation as compared to 1875nm. While radiant exposures remained relatively constant across different spot sizes when using 1450nm, the threshold radiant exposures for 1875nm exposures increased with spot size suggesting deeper nerves fibers tend to be activated. Moreover, exposures using a flat-top beam profile yielded less variability in the stimulation threshold than those using a Gaussian profile. As in previous studies, histology confirmed that damaging radiant exposures are several times greater than the stimulation threshold for both 1450nm and 1875nm. Our results provide valuable insight for future studies involving INS and for further developing INS as both a research and clinical tool.

Ultrasound has been recently explored as a new modality for neural modulation. However, one of the challenges in ultrasound neural modulation is that delivery of transcranial ultrasound would inevitably go through the skull, and eventually reach the cochlear through bone transduction. Moreover, the presence of skull will compromise ultrasound focus, resulting in poor spatial resolution. Here, we developed a miniaturized Fiber-Optoacoustic Converter (FOC), which has a diameter of 600 μm, and can convert nano-second laser pulses into omni-directional acoustic waves through the optoacoustic effect. The ball shaped FOC is composed of one ZnO /epoxy based diffusion layer and two graphite/epoxy based absorption layer. The radiofrequency spectrum of the generated US frequency ranges from 0.1-5 MHz, with multiple frequencies peaks at 0.5, 1 and 3MHz. Using this FOC system, we show that ultrasound can directly activate individual cortical neuron in vitro, and generate intracellular Ca2+ transient without neural damage. We next demonstrate that the FOC is activates neurons with a radius of 500 μm around the FOC tip, delivering superior spatial resolution. The stimulation effect is specific to neurons, but not glial cells. We also provide evidence of transient mechanical disturbance of neuronal membrane as the mechanism for FOC neural modulation. Finally, we combine FOC neural modulation with electrophysiology, and achieve direct and spatially confined neural stimulation in vivo.

Vagus nerve interfacing is of interest due to its central role in parasympathetic regulation of the visceral organs, as well as its modulatory effects on the brain, which have been shown to influence epilepsy, depression and migraines. Electrical vagus nerve stimulation (VNS) has shown therapeutic effect in humans, yet it lacks the specificity for controlling and studying targeted pathways. In contrast, optical techniques may enable axon-specific neuromodulation using genetically targeted opsin expression and spatial patterning of the photo-stimulus. In addition to light-activated stimulation, calcium-sensitive fluorescent reporters such as GCaMP6 present a pathway for axon-specific optical recording of activity. We demonstrate in vivo photo-stimulation and two-photon GCaMP6 fluorescence imaging in the vagus nerve using a novel GRIN lens-coupled nerve cuff in the anesthetized mouse. A pulsed near-IR laser (1040 nm, 300 fs) was modified by a spatial light modulator (SLM) in the Fourier plane and focused by the microscope objective through a GRIN relay lens to the cervical vagus nerve. By actuating the SLM, spatially selected regions of axons could be differentially stimulated within the nerve. Mouse vitals were monitored with a MouseOx suite and used to detect physiological changes in response to photo-stimulation. We were able to induce differential modulations in heart rate, respiratory rate, and blood-oxygen saturation upon photo-stimulation of selective spatial regions of the nerve. Additionally, we recorded two-photon GCaMP6 Ca²⁺ transients in vagal axons in response to both photo-stimulation and electrical stimulation.

In recent years, brain research has made a lot of progress with the help of new techniques such as fMRI. Most insights were gained on animal models and thus have a limited applicability to human subjects. The generation and application of human stem-cell-derived functional neural circuits therefore promises novel insights, e.g. into neurodegenerative diseases. These networks are often studied in vitro using multielectrode arrays for electrical stimulation and recording, often at low spatial resolution and specificity. Optogenetics allows for the functional control of genetically altered cells with light stimuli at high spatiotemporal resolution. Current optogenetic investigations of human neural networks are often conducted using full field illumination, potentially masking important functional information. To circumvent this, we built a holographic beam shaping setup using a fast ferroelectric spatial light modulator for single cell optogenetic stimulation, which we presented previously [1]. We achieve a lateral resolution of 8 µm in a field of view of 1.5x1.5 mm at a maximum temporal resolution of 0.6 ms using binary Fresnel holograms. In the presentation, we will first discuss our setup. Then, we will present our experiments and results on the spatiotemporal investigation of iPSC-derived human neural networks expressing wild-type channelrhodopsin-2 [2]. [1] F. Schmieder, M. Henning, L. Büttner, S. Klapper, K. Lenk, V. Busskamp, J. Czarske, “Targeted optogenetic investigation of in vitro human iPSC-derived neuronal networks”, SPIE Photonics West, 27.01.-01.02.2018, San Francisco, USA [2] Klapper, S. D.; Sauter, E. J.; Swiersy, A.; Hyman, M. A. E.; Bamann, C.; Bamberg, E.; Busskamp, V. On-demand optogenetic activation of human stem-cell-derived neurons. Sci. Rep. 2017, 7, 14450

Neuromodulation has the potential to treat various diseases (i.e., heart failure, obesity). Several clinical trials have recently failed because of the inability to modulate small-diameter fibers. Previously, we demonstrated preferential inhibition of small-diameter fibers using infrared neuromodulation (IRN). To understand the mechanism of action, we did a mathematical analysis which suggested that any modality acting primarily on the axonal surface would preferentially affect small-diameter axons. To test our hypothesis, we examined whether isotonic glucose solution would give results similar to IRN. We stimulated the left and right pleural-abdominal connective nerves of Aplysia californica and recorded the resulting compound action potentials (CAPs). We designed a chamber with three isolated compartments through which the nerve passes sequentially. Aplysia saline is perfused in the two outer chambers while the middle chamber can be perfused with either Aplysia saline or an isotonic glucose solution (10.21 w/v %). The width of the middle chamber is adjustable to vary the length of nerve perfused by the isotonic glucose solution. As the length of the middle chamber increases, recorded CAPs are initially unaffected, then show a loss of peaks representing small-diameter axons, then show no activity. We can restore full, unchanged CAPs by washing out the glucose solution and replacing it with Aplysia saline. These results support the hypothesis that any modality (e.g., both IRN and isotonic glucose solution) acting primarily on the axonal surface would preferentially affect small-diameter axons. Future studies will compare IRN with isotonic glucose block.

The ability to quickly recover to a resting heart rate after physical exertion is an indicator of heart health. Experiments with humans and other mammals use exercise to induce physiological distress and to increase the heart rate in subjects. In Drosophila models, it is not feasible to induce increased physical activity while simultaneously recording their heartrate. Cardiac pacing can be used as an analog to physical activity because it forces the fly heart to follow a pacing rate which induces physiological distress. In the established Drosophila models, we used non-invasive red light optogenetic pacing to activate or inhibit physical activities while simultaneously record the heart rate (HR) using the optical coherence microscopy technique. In ReaChR flies, a recovery period with a gradually increasing HR was observed after inducing tachycardia through the red light stimulation. The maximum HR and the time period before reaching the resting heart rate after pacing ceases were studied in the fruit flies. Physiological distress was also induced by reducing or halting the NpHR Drosophila’s heart rate through red light stimuli. After induced bradycardia pacing and cardiac arrest a recovery period of rapid heart beating was observed. The cardiac recovery after pacing could be extensively used as an indicator in understanding the correlation of age with cardiac deterioration in different animal models and humans.