This PDF file contains the front matter associated with SPIE Proceedings Volume 10482, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

To enable the understanding and repair of complex biological systems such as the brain, we are creating novel optical tools that enable molecular-resolution maps of large scale systems, as well as technologies for observing and controlling high-speed physiological dynamics in such systems. First, we have developed a method for imaging large 3-D specimens with nanoscale precision, by embedding them in a swellable polymer, homogenizing their mechanical properties, and exposing them to water - which causes them to expand isotropically manyfold. This method, which we call expansion microscopy (ExM), enables scalable, inexpensive diffraction-limited microscopes to do large-volume nanoscopy, in a multiplexed fashion. Second, we have developed a set of genetically-encoded reagents, known as optogenetic tools, that when expressed in specific neurons, enable their electrical activities to be precisely driven or silenced in response to millisecond timescale pulses of light. Finally, we are developing novel reagents and systems to enable the imaging of fast physiological processes in 3-D with millisecond precision. In this way we aim to enable the systematic mapping, control, and dynamical observation of complex biological systems like the brain.

Optogenetic modulation of neural circuits has opened new avenues into neuroscience research, allowing the control of cellular activity of genetically specified cell types. Optogenetics is still underdeveloped in the peripheral nervous system, yet there are many applications related to sensorimotor function, pain and nerve injury that would be of great benefit. We recently established a method for non-invasive, transdermal optogenetic stimulation of the facial muscles that control whisker movements in mice (Park et al., 2016, eLife, e14140)1. Here we present results comparing the effects of optogenetic stimulation of whisker movements in mice that express channelrhodopsin-2 (ChR2) selectively in either the facial motor nerve (ChAT-ChR2 mice) or muscle (Emx1-ChR2 or ACTA1-ChR2 mice). We tracked changes in nerve and muscle function before and up to 14 days after nerve transection. Optogenetic 460 nm transdermal stimulation of the distal cut nerve showed that nerve degeneration progresses rapidly over 24 hours. In contrast, the whisker movements evoked by optogenetic muscle stimulation were up-regulated after denervation, including increased maximum protraction amplitude, increased sensitivity to low-intensity stimuli, and more sustained muscle contractions (reduced adaptation). Our results indicate that peripheral optogenetic stimulation is a promising technique for probing the timecourse of functional changes of both nerve and muscle, and holds potential for restoring movement after paralysis induced by nerve damage or motoneuron degeneration.

In mammals, neurons are accompanied by numerous cell types, including microglia, oligodendrocytes, and astrocytes. These cells work cohesively to stabilize the neuronal microenvironments and ensure cellular functionality is unperturbed. Studies involving infrared neural stimulation (INS) have been focused on neuronal-type cellular responses, while the effects on non-neuronal cells have been largely unexplored. Astrocytes, in particular, play a key role in maintaining extracellular ion and neurotransmitter concentrations, maintaining osmotic balance, preserving synaptic connectivity, and in mediating neurovascular coupling responses in the brain. Previous work has speculated whether astrocytes are affected by INS in the brain. Here, we characterize the effect of INS on astrocytes using calcium dynamics in response to varying optical parameters and explore whether astrocytic response is driven by neuronal activity or directly by transient thermal energy deposition. Using video-rate calcium-fluorescence multiphoton imaging and nonlinear microscopy of cultured cells, we found that calcium activity in astrocytes is sensitive to infrared light independent of neuronal presence. These cells exhibited similar time-dependent behavior as reported in previous in vivo studies. Success in eliciting calcium responses from a cell, as well as response amplitude, appears to correlate with radiant exposure. The duration and shape of such responses are variable from cell to cell, with some responses aligning with previous observations. Our results suggest that collateral activation of non-neuronal cells, particularly in astrocytes, may play a role when applying INS in vivo.

Recently, optogenetics has provided interesting insights into cardiovascular research, leading to cardiac pacing, re-synchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac pathway manipulation, optical stimulation has not been directly driven by the cardiac electrical dynamics, limiting the full potential of such a new technology. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control whole mouse heart electrical activity. The system combines an ultrafast wide-field mesoscope with a digital micro-mirror device (Texas Instruments Lightcrafter), capable of drawing arbitrarily-chosen patterns, thus allowing optogenetic activation. Cardiac functionality can be manipulated either in free-run mode with sub-millisecond temporal resolution or in a closed-loop fashion: an ad hoc hardware and software platform allows real-time intervention capable of reacting to threatening anomalous electrical conditions within 2 ms. The methodology has been applied to restore atrioventricular block, by triggering the optical stimulation of the ventricle according to optically mapped atrial activity. Furthermore, real-time intra-ventricular manipulation of the propagating electrical wave-front has been demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. The development of this innovative optical methodology provides the first proof-of-concept that a real-time, self-sustaining, optical-based stimulation can efficiently control cardiac rhythm in normal and abnormal conditions, promising a new approach to the investigation of (patho)physiology of the heart.

In cardiac optogenetics, cardiac functions of animals such as rat, zebrafish, and fruit fly, are controlled through optical excitation of opsin expressed cardiac tissues. In the last few years, this non-invasive cardiac control method has been developed rapidly as an alternative to the traditional technique of electrical stimulation. However, the strong absorption and scattering of the excitation light needed for commonly used opsins limit the optical penetration depth in tissue, which hampers the development of cardiac optogenetics. In this work, we express red-shifted opsins (ReaChR and halorhodopsin) in the heart of the established Drosophila melanogaster model,and use red-light stimulation for deep penetration of excitation light into the myocardial structures. Mmode images acquired through optical coherence microscopy (OCM) imaging demonstrate controlled heart function in vivo and in real time throughout the life cycle of Drosophila. Fast kinetics, high safety and high heart-rate adjustability were shown with short pulse width, low excitation power density, and wide frequency tuning range, respectively, in the pacing study. Stimulation power was also tuned to characterize the optimal excitation power densities for reliable cardiac function inactivation, which were proved safe for each developmental stage. Both groups of flies exhibited high cardiac stimulation efficiencies. This study demonstrates non-invasive cardiac control through activating and inhibiting heart functions of an intact animal, which is promising for scientific study and clinical treatment of cardiac diseases, such as congenital or posteriority bradycardia, tachycardia, and regional mechanical dys-synchrony.

Detailed understanding of mechanisms and instabilities underlying the onset, perpetuation, and control of cardiac arrhythmias is required for the development, further optimization, and translation of clinically applicable defibrillation methods. Recently, the potential use of optogenetic tools using structured illumination to control cardiac arrhythmia has been successfully demonstrated and photostimulation turned out to be a promising experimental tool to investigate the dynamics and mechanisms of multi-site pacing strategies for low-energy defibrillation. In order to study the relation between trigger and control mechanisms of arrhythmic cardiac conditions without external affecting factors like eventually damaging fiber poking, it is important to establish a non-invasive photostimulation method. Hence, we applied a custom-configured digital light processing micromirror array operated by a high-speed FPGA, which guarantees a high frequency control of stimulation patterns. The integration into a highly sophisticated optical experiment setup allows us to record photostimulation effects and to proof the light pulse as origin of cardiac excitation. Experiments with transgenic murine hearts demonstrate the successful induction and termination of cardiac dysrhythmia using light crafting tools. However, the complex spatiotemporal dynamics underlying arrhythmia critically depends on the ratio of the characteristic wavelength of arrhythmia and substrate size. Based on the experimental evidence regarding the feasibility of optical defibrillation in small mammals, the transfer in clinically relevant large animal models would be the next milestone to therapeutic translation. Thus, the presented experimental results of optogenetically modified murine hearts function as originator for ongoing studies involving principle design studies for therapeutic applicable optical defibrillation.

Brain functions and related psychiatric disorders have been investigated by recording electrophysiological field potential. When recording it, a conventional method requires fiber-based apparatus connected to the brain, which however hampers the simultaneous measurement in multiple animals (e.g. by a tangle of fibers). Here, we propose a fiber-free recording technique in conjunction with a ratiometric bioluminescent voltage indicator. Our method allows investigation of electrophysiological filed potential dynamics in multiple freely behaving animals simultaneously over a long time period. Therefore, this fiber-free technique opens up the way to investigate a new mechanism of brain function that governs social behaviors and animal-to-animal interaction.

Monitoring of visual functioning of the retina is significant for characterizing retinal degenerative diseases. Electroretinogram is the current method for measuring the electrical responses of the retina to light. However, it requires placement of electrodes on cornea, leading to contact related uncomfortable feeling. Here, we report use of near-infrared low-coherent light for non-contact, label-free in-vivo detection of retinal activities in response to visual stimulation. We utilized phase sensitive optical coherence tomography for measuring fluctuations of light reflected from retina of wild type and retinal degenerated mice. With visual stimulation, fluctuations in optical path length difference were found to be higher than that without visual stimulation in wild type mice. However, no such changes observed in mice with photoreceptor degeneration. Our findings open up possibility for clinical use of this method for non-contact label free characterization of retinal functioning and identification of dystrophies.

Though primary visual cortex is known to maintain its retinotopy in subjects with retinal degeneration despite prolonged visual loss, detailed knowledge of how optogenetic sensitization of higher order neurons manifests in restoration of visual cortical activity is currently lacking. Here, we report development and characterization of bioluminescent opsin for simultaneous optical modulation and imaging of retinal and cortical activities using spectrally separated activation and detection bands. This new bioluminescent technique does not require an additional phototoxic external excitation source (as used for fluorescence). We quantified changes in bioluminescence activities in visual cortex of mice upon visual stimulation of the retina. The observed increased neural activities were found to correlate with the visual stimulation patterns. This method will be useful for monitoring changes in visual cortical activities during progression and repair of retinal degenerative diseases. Further, with integration of stimulation source, we envision development of a modular and scalable interface system with the capability to serve a multiplicity of applications to modulate and monitor large-scale activity in the nervous system.

Optogenetics is a powerful tool for neural control, but controlled light delivery beyond the superficial structures of the brain remains a challenge. For this, we have developed an optrode array, which can be used for optogenetic stimulation of the deep layers of the cortex. The device consists of a 10×10 array of penetrating optical waveguides, which are predefined using BOROFLOAT® wafer dicing. A wet etch step is then used to achieve the desired final optrode dimensions, followed by heat treatment to smoothen the edges and the surface. The major challenge that we have addressed is delivering light through individual waveguides in a controlled and efficient fashion. Simply coupling the waveguides in the optrode array to a separately-fabricated μLED array leads to low coupling efficiency and significant light scattering in the optrode backplane and crosstalk to adjacent optrodes due to the large mismatch between the μLED and waveguide numerical aperture and the working distance between them. We mitigate stray light by reducing the thickness of the glass backplane and adding a silicon interposer layer with optical vias connecting the μLEDs to the optrodes. The interposer additionally provides mechanical stability required by very thin backplanes, while restricting the unwanted spread of light. Initial testing of light output from the optrodes confirms intensity levels sufficient for optogenetic neural activation. These results pave the way for future work, which will focus on optimization of light coupling and adding recording electrodes to each optrode shank to create a bidirectional optoelectronic interface.

Light-based therapies have been established for various indications, such as skin conditions, cancer or neonatal jaundice. Advances in the field of optogenetics open up new horizons for light-tissue interactions with an organism-wide impact. Excitable tissues, such as nerve and muscle tissues, can be controlled by light after the introduction of light-sensitive ion channels. Since these organs are generally not easily accessible to illumination in vivo, there is an increasing need for effective biocompatible waveguides for light delivery. These devices not only have to guide and distribute the light as desired with minimal losses, they should also mimic the mechanical properties of the surrounding tissue to ensure compatibility. In this project, we are tuning the properties of hydrogels from poly(ethylene glycol) derivatives to achieve compatibility with muscle tissue as well as optimal light guiding and distribution for optogenetic applications at the heart. The excitation light is coupled into the hydrogel with a biocompatible fiber. Properties of the hydrogel are mainly tuned by monomer length and concentration. Total reflection can be achieved by embedding a fiber-like hydrogel with a high refractive index into a second, low refractive index gel. Different geometries and scattering microparticles are used for light distribution in a flat gel patch. Targeted cell attachment can be achieved by introducing a protein layer to the otherwise bioinert gel. After optimization, the hydrogel may be used to deliver light for the excitation of genetically altered cardiomyocytes for controlled contraction.

In recent years, numerous methods have been sought for developing novel solutions to counter neurodegenerative diseases. An objective that is being investigated by researchers is to develop cortical implants that are able to wirelessly stimulate neurons at the single cell level. This is a major development compared to current solutions that use electrodes, which are only able to target a population of neurons, or optogenetics, which requires optical fiber-leads to be embedded deep into the brain. In this direction, the concept of wireless optogenetic nanonetworks has been recently introduced. In such architecture, miniature devices are implanted in the cortex for neuronal stimulation through optogenetics. One of the aspects that will determine the topology and performance of wireless optogenetic nanonetworks is related to light propagation in genetically-engineered neurons. In this paper, a channel model that captures the peculiarities of light propagation in neurons is developed. First, the light propagation behavior using the modified Beer-Lambert law is analyzed based on the photon transport through the nervous tissue. This includes analyzing the scattering light diffraction and diffusive reflection that results from the absorption of neural cell chromophores, as well as validating the results by means of extensive multiphysics simulations. Then, analysis is conducted on the path loss through cells at different layers of the cortex by taking into account the multi-path phenomenon. Results show that there is a light focusing effect in the soma of neurons that can potentially help the to stimulate the target cells.

The efficient and targeted delivery of genes and other impermeable therapeutic molecules into retinal cells is of immense importance for therapy of various visual disorders. Traditional methods for gene delivery require viral transfection, or use of physical and chemical methods which suffer from one or many drawbacks such as invasiveness, low efficiency, lack of spatially-targeted delivery, and can generally have deleterious effects such as unexpected inflammatory responses and immunological reactions. Further, for effective dry-age related macular degeneration (dry-AMD) therapy involving geographic atrophies of the retina, it requires to localize the delivery of the targeted opsin-encoding genes to specific retinal cells in atrophied-regions. Here, we report near-infrared laser based Nano-enhanced Optical Delivery (NOD) of opsin-encoding genes into retina of mouse models of retina degeneration in-vivo. In this method, the field enhancement by gold nanorods is utilized to transiently perforate retinal cell membrane to deliver exogenous molecules to cells in the targeted area of retina. SDOCT was used to monitor if there is any damage to retina and other ocular structures. The expression and functioning of opsin in targeted retina after in-vivo NOD in the mice models of retinal degeneration opens new vista for re-photosensitizing retinas with geographic atrophies in dry-AMD.

Holographic optogenetics is an emerging tool for distributed control of spatiotemporal neuronal activity. Establishing causality between specific sequences of neuronal activity and behavior requires manipulating this code at the level of individual neurons while recording neural responses and behavioral readout. However, traditional methods of optogenetic perturbation lack the ability to emulate natural patterns of neural activity, or to rapidly alter the activity of specific neurons deep in the brain. To address the goal of producing behaviorally relevant sequences of neural activity, we have developed an all-optical, rapid two-photon optogenetic stimulation and imaging system with cellular resolution and 5 ms temporal precision. Using an amplified laser with high peak pulse power, together with wavefront shaping methods using a fast spatial light modulator (3 ms switching time), we were able to stimulate dozens of neurons deep in the olfactory bulb (>350 µm) at a high rate (>100 Hz) with cellular resolution. We optimized the system parameters to enable efficient excitation with a low power budget, to enable the simultaneous stimulation of many cells (~60). We then demonstrated stimulation of mitral and tufted cells, the projection neurons of the olfactory bulb, at a high rate, generating artificial odor-evoked responses. We will present the system characteristics and discuss its potential applications for manipulating and reading neuronal activity on a behaviorally relevant spatiotemporal scale to dissect the activity codes that guide behavior across different modalities.

In the context of near-infrared neurostimulation, we report on an experimental hybrid electrode allowing for simultaneous photonic or electrical neurostimulation and for electrical recording of evoked action potentials. The electrode includes three contacts and one optrode. The optrode is an opening in the cuff through which the tip of an optical fibre is held close to the epineurium. Two contacts provide action potential recording. The remaining contact, together with a remote subcutaneous electrode, is used for electric stimulation which allows periodical assessment of the viability of the nerve during the experiment. A 1470 nm light source was used to stimulate a mouse sciatic nerve. Neural action potentials were not successfully recorded because of the electrical noise so muscular activity was used to reflect the motor fibres stimulation. A recruitment curve was obtained by stimulating with photonic pulses of same power and increasing duration and recording the evoked muscular action potentials. Motor fibres can be recruited with radiant exposures between 0.05 and 0.23 J/cm² for pulses in the 100 to 500 μs range. Successful stimulation at short duration and at a commercial wavelength is encouraging in the prospect of miniaturisation and practical applications. Motor fibres recruitment curve is a first step in an ongoing research work. Neural action potential acquisition will be improved, with aim to shed light on the mechanism of action potential initiation under photonic stimulation.

Multifunctional fibers are developed worldwide for enabling many new advanced applications. Among the multiple new functionalities that such fibers can offer according to their design, chemical composition and materials combination, the co-transmission of light and electrical signals is of first interest for sensing applications, in particular for optogenetics and electrophysiology. Multifunctional fibers offer an all-solid approach relying on new ionic conducting glasses for the design and manufacturing of next generation optrodes, which represents a tremendous upgrade compared to conventional techniques that requires the utilization of liquid electrolytes to carry the electrical signal generated by genetically encoded neuronal gated ion channels after optical excitation. After a systematic study conducted on different ion-conductive glass systems, silver phosphate-based glasses belonging to the AgI-AgPO₃-WO₃ and AgI−AgPO₃−Ag₂WO₄ systems were found to be very promising materials for the target application. Several types of fibers, including single-core step-index fibers, multimaterial fibers made of inorganic and optical polymeric glasses have been then fabricated and characterized. Light transmission ranging from 400 to 1000 nm and electrical conductivity ranging from 10⁻³ and 10⁻¹ S·cm−1 at room temperature (AC frequencies from 1 Hz to 1 MHz) were demonstrated with these fibers. Very sharp fiber tapers were then produced with high repeatability by using a CO₂ laser optical setup, allowing a significant shrinking from the fiber (300 μm diameter) to the taper tip (25-30 μm diameter).

Infrared light can be used to modulate the activity of neuronal cells with broad generality and without any need for exogenous materials. The action potential response has been shown to be associated with heating due to the absorption of light by water in and around the illuminated tissues, which gives rise to at least two distinct processes: namely, the temperature pulses cause depolarizing capacitive currents due to an intramembrane thermo-mechanical effect, and in addition, temperature-sensitive TRPV ion channels (and likely, voltage-gated channels) drive additional membrane depolarization. However, substantial differences between the activation threshold of primary auditory neurons (<20 mJ/cm^2) and other neuronal types (>300 mJ/cm^2) in vivo have generated some controversy in the field. A temperature-dependent Hodgkin-Huxley type model, which combines capacitive currents and the experimentally-derived characteristics of voltage-gated potassium and sodium ion channels in primary auditory neurons, was used to accurately explain the in vitro response to 1870 nm infrared illumination. TRPV channels do not make a significant contribution in this case, suggesting that the detailed mechanism of the neuronal response to infrared light is dependent on the specific cell type. Furthermore, based on this detailed understanding of the cell behaviour, it is shown that action potentials cannot be generated at safe laser power levels. This suggests that the previously reported response of the auditory system to infrared stimulation in vivo might arise from a different mechanism, and calls into question the potential usefulness of the effect for auditory prostheses.

All-optical systems for stimulating and imaging neuronal activity have served as powerful tools for understanding the underlying circuitry of the brain. Experiments using these setups, however, tend to choose stimulation locations based solely on what brain regions are of interest, and take for granted that stimulation effects may vary even within localized brain regions. We thus have developed an algorithm for acquiring neuronal activity via calcium imaging data to assess network connectivity. These parameters include the signal rise time, decay time, inter-event intervals, and the timing and amplitude of signal peaks. These parameters are then compared between cell clusters for similarities, and used as a basis for establishing interconnectivity. Additionally, we have incorporated both temporal and spatial correlation functions to assess inter-neuronal connectivity based on these parameters. This data is then run through a genetic algorithm, applying weights to cells with similar parameters to learn which are interconnected in a given field-of-view. For this study, hippocampal neurons extracted from 2 day old transgenic mice (GCaMP6s, Jackson Labs), - cultured for 2 weeks and imaged under single and two-photon conditions. Single-photon imaging was performed under a commercial Zeiss microscope, whereas two-photon imaging was performed with an in-house imaging system. Results demonstrate a strong correlation between these parameters and cellular connectivity, making them noteworthy markers for targeted stimulation. This study demonstrates an efficient method of assessing network connectivity for various imaging techniques, and hence directed targeting for optogenetic stimulation.