This PDF file contains the front matter associated with SPIE Proceedings Volume 11227 including the Title Page, Copyright information, Table of Contents, Introduction, and Conference Committee listing.

Understanding and repairing complex biological systems, such as the brain, requires new technologies that enable such systems to be observed and controlled with great precision, across extended spatial and temporal scales. We are discovering new molecular principles that are leading to such technologies. For example, we recently discovered that it was possible to physically magnify biological specimens manyfold, in an even way, by embedding them in dense swellable polymers, mechanically homogenizing the specimens, and then adding water to isotropically swell the specimens. In this method, which we call expansion microscopy (ExM), we enable scalable, inexpensive diffraction-limited microscopes to do large-volume nanoscopy, in a multiplexed fashion – important, for example, for brain mapping. As another example, we discovered that microbial opsins, genetically expressed in neurons, could enable their electrical activities to be precisely driven or silenced in response to millisecond timescale pulses of light. These tools, called optogenetic tools, are enabling causal assessment of the contribution of defined neurons to behaviors and pathologies in a wide variety of basic science settings. Finally, we have developed new methods of directed evolution, and discovered mutant forms of optogenetic tools that enable precision fluorescent imaging of the high-speed voltage of neurons in the living brain. We share all these tools freely, and aim to integrate the use of these tools so as to lead to comprehensive understandings of neural circuits.

Understanding the central nervous system and the human brain in particular represents one of the most challenging endeavors in current science. This interest is not only driven by the need to find new methods for curing neural disorders. Increasing efforts are dedicated to the development of hardware mimics of the nervous system in the form of artificial neural networks for computational purposes, which also reflects substantial interest in a deeper understanding of the brain’s functionality. Here, we present a nanophotonic approach to enable a precise investigation of neural activity at the level of a single synapse. Key component is an integrated photonic chip, which serves as a platform for neural growth. Our simulations show, that an optogenetically modified nerve cell in close vicinity to a photonic waveguide can be efficiently stimulated by the waveguide’s evanescent field, even with a low optical input power in the order of microwatts. These results pave the way for designing a versatile analysis tool to investigate both the behavior of individual neurons and their synergistic function in a neural network at the same time.

The ability to both induce and monitor neural activity at cellular resolution is necessary to comprehend the activation paradigm in sensory processing and for the development of effective neuromodulation treatments. Our lab has produced an actuator-sensor construct via the hybridization of an optogenetic actuator (bMCOII) and bioluminescent Ca²⁺ sensor (GeNL) which allows continuous monitoring of neural activity with high spatiotemporal resolution. Modeling revealed that the construct is bound to the cell membrane through 14 transmembrane helices with the Ca²⁺ -bioluminescence indicator domain 20-40 Å inside the cytoplasm (membrane localization confirmed by imaging). The construct can be used to stimulate neural activity with very low intensity (10 μW/mm² ) light but unlike fluorescent methods, requires no excitation light. Activation of the opsin causes influx of Ca²⁺ by opening MCOII-channels via trans-cis isomerization of all-trans-retinal. When these ions bind the indicator domain there is an increase in bioluminescence intensity. We observed significant correlation between the magnitude and kinetics of induced electrical activities and Ca²⁺ -bioluminescence. Longterm (>14 hours) recording of evoked neural activity in the visual cortices of murine models allowed the quantification of the strength of sensory activation. Additionally, through Artificial Intelligence-based Neural Activation Parameters, the Ca²⁺ -bioluminescence signals were used to map network activity patterns. We also observed delayed, secondary Ca²⁺ - bioluminescence responses murine visual cortices. These may be astrocyte-mediated responses to direct optogenetic and indirect visual stimulation. Our technique will enable the development of a scalable, and modular interface system which can be expanded to monitor and modulate a variety of neurological activities.

Decades of studies in non-human primates (NHPs) have been conducted in conditions of strict experimental control of perception and behavior in order to understand the functions of the brain. For instance, experiments focused on visual perception often involve head fixation, unnaturally long eye fixation periods, and attention to locations away from the center of gaze. These paradigms require long training periods (months to a year). While such studies have advanced our knowledge of brain function tremendously, a recurring question is how relevant the gathered data are to natural behavior. The availability of cutting-edge technologies and computational power can free us from the limitations of classical experimental paradigms and usher in a new generation of neuroscience questions focused on how the brain functions in real world settings. Here we demonstrate a small head-mounted multimodal device for stimulating and recording domain-based cortical activity in NHPs. This device consists of an intrinsic optical signal imaging camera and a wireless LED stimulator. To evaluate the functionality of this device, we compared its imaging performance in vivo to that of current benchtop intrinsic imaging systems and demonstrated the efficacy of wireless optogenetic stimulation while conducting awake intrinsic imaging. Our device device has been constructed from off the shelf components and offers multiareal and targeted stimulation and recording capabilities in minimally restrained subjects. It has a potential to contribute to our understanding of cortical encoding of perception and behavior and could have clinical relevance for the development of brain-machine interfaces.

Optogenetics is a powerful tool for neural control that supports perturbation of specific cell types. The Utah Optrode Array, paired with a µLED array, is an optical stimulation device for precise multi-site light delivery in deep cortical layers. We describe recent developments in the evolution of this device, including methods for improved light coupling and device encapsulation.

In the past decade, in vivo models for optogenetic applications have gained importance, especially in the fields of cardiology and neuroscience. To reliably evoke the desired reactions while minimizing adverse effects, the stimulation power must be carefully adjusted. The relevant light intensity for cells in deeper layers of scattering tissue is not easily extrapolated from the power threshold of single cells. In this study, we evaluated a model for deep tissue optogenetic stimulation, using a heart-like cell line and tissue phantoms. Phantoms were fabricated from PDMS and titanium dioxide particles and possessed highly reproducible optical properties. Scattering and absorption coefficients were modeled to match those of realistic tissues. Since power of light traveling through tissues decays exponentially with respect to the scattering and absorption coefficients, the required input power was expected to increase exponentially by the same factor. To test this hypothesis, cells were stimulated through tissue phantoms of varying thickness with different modes of illumination. Cellular reactions revealed that the simplified assumptions were not sufficient to predict the input power required to reach the stimulation threshold. We provide a more comprehensive model to assess cellular reactions in scattering tissues a priori. This study has implications for the use of optogenetics in tissue models, organs and in vivo models as the outcomes can be transferred to different types of cells and tissues.

Optogenetics integrated with a variety of genetically encoded methods have been considered a very powerful technique for identifying neurovascular coupling mechanism. Recently, optogenetics has been applied to non-neuronal cells as well as neurons, to study the functions of various organs and tissues such as cardiovascular and bladder. However, the cerebrovascular network has a complicated environment in which cells and tissues with various features and functions coexist. In order to successfully apply optogenetics to study neurovascular coupling mechanism, it is necessary to develop not only optical stimulation techniques but also appropriate monitoring techniques. In addition, it has been very difficult to develop an appropriate optical system, since it is necessary to operate with the neurovascular network of a living animal maintained in the normal condition. Therefore, we have developed a fiber-based all-optical system that enables successful application of optogenetics to study neurovascular coupling mechanisms in living mouse. The developed optical system can perform rapid fluorescence imaging and optical stimulation at the same time, so it can measure various cell specific reactions and rapid blood flow that change after optical stimulation. In this study, we were able to observe changes in blood vessel diameter and blood flows caused by the cell activity of smooth muscle cells after selective optical stimulation using the developed all-optical system in SM22α-ChR2 transgenic mice. Also, the developed all-optical system can be used to study cerebrovascular disease or hemodynamics using optogenetics in vivo.

Optogenetics is a valuable tool set for the investigation of neuronal connections. Data on the connectome of human neurons is indispensable e.g. for the understanding of human neurodegenerative diseases. Providing this data for in vitro human stem-cell derived neuronal networks is the aim of our research. We use a holographic illumination setup with up to 1.7 kHz frame rate, 8 µm spatial resolution and 31 mm working distance for the stimulation of neurons expressing Channelrhodopsin2. In this work, we will present the measured functional connectivity of single neurons we retrieve using spatially resolved stimulation and simultaneous extracellular multi electrode recording.

Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light modulated protein stabilization system (GLIMPSe) to control intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins, phosphatases and kinases. Kinetics study showed that light-induced protein stabilization could be achieved within 30 minutes of blue light stimulation. Overall, GLIMPSe enables target-independent optogenetic control of protein activities, minimizes the systematic variation embedded within different photoactivatable proteins and promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.

Recent development of light-controlled cellular activity, known as optogenetics, has sparked interest in new methods for neural and cellular activation. Since its inception, optogenetics has impacted neuroscience research in many ways providing new insight into the functioning of neuronal circuitry. Even though great strides have been made in this community, there is still much to be discovered. Therefore, there is a need for improvements and innovative capabilities for light delivery and control. Although there are many avenues to overcome these limitations, we propose the use of non-traditional optogenetic wavelengths in order to move towards a non-invasive optogenetic approach. Alternatives to optogenetics techniques have been developed and coined “thermogenetics,” “magnetogenetics,” and “radiogenetics.” For example, the use of radio frequencies (RFs) to drive cellular stimulation has been shown to provide a big advantage in remote modulation of signaling pathways because it is non-invasive, easily adaptable and does not require the insertion of electrodes. Thus, our goal was to branch out from traditional optogenetics and focus on the mechanistic properties of RFs on proteins and cellular pathways in order to drive the future use of non-traditional optogenetics. In this work, we proposed to synthetically generate a new sensing element to enable its potential modulation by RF fields. The ability to remotely modulate the expression of novel sensing proteins would provide a tool to better understand the human neuronal network as well as develop a technique towards the improvement of human performance.

Herein, we report use of near-infrared low-coherent light for non-contact, label-free in-vivo detection of retinal activity in response to visual illumination. Our multifractal phase-OCT employ phase/multifractal analysis to decipher layer specific cellular activity during visual stimulation to assess the functional state of retina. Our OCT-based interferometric technique coupled with in-depth multifractal analysis differentiated retinal activities between wild-type and mice with retinal dystrophy. Our findings open up possibility of clinical translation of multifractal phase-OCT for non-contact label free evaluation of retina health, progression of retinal dystrophies, and as well as for monitoring functional recovery after therapy.

Infrared neural inhibition (INI) is a method of blocking the generation or propagation of neural action potentials through heating with infrared lasers that target peaks in the water absorption spectrum (1460 nm, 1875 nm). Recent work showed that the length of axon heated modulates the temperature needed for inhibition. Different irradiation lengths for INI were tested in Aplysia nerves (n=7). Once 1 mm of irradiation was exceeded, longer irradiation lengths resulted in higher temperatures at inhibition threshold. Optimizing irradiation lengths of nerve to minimize temperatures will help inform the design of devices for longitudinal animal studies and human implementation.

We have shown previously that astrocytes are independently sensitive to IR exposure in the context of infrared neural stimulation (INS). More recently, our advances have identified some physiological basis of astrocytic sensitivity to INS. Single pulse INS evokes intracellular calcium signaling primarily mediated through inositol-triphosphate receptor (IP¬3R) activation, rather than thermally sensitive transient receptor protein (TRP) pathways. Interestingly, different dosing strategies for IR appear to evoke different physiology. Our findings suggest that tailoring irradiation parameters with INS may be used for preferentially drive separate physiological responses in astrocytes, and possibly other cell types.

Infrared neural inhibition (INI) is a relatively new modality of neural control which has potential as a novel pain therapy due to its high spatial specificity and selective inhibition of small diameter neurons at lower temperatures. Computational modeling using a modified Hodgkin-Huxley model in the squid giant axon has shown that temperature sensitive potassium currents mediate INI’s thermal block. This model was modified to reflect Aplysia parameters which have smaller unmyelinated axons on the order of mammalian C fibers, and simulated results were validated in vitro. Results support he hypothesis that potassium currents are needed to create a thermal block.

Infrared neural stimulation (INS) has the potential to serve as a spatially precise tool for neuroscientists and clinicians. Because of difficulties comparing various INS techniques in the current literature, we present a study comparing the stimulation threshold and transition rate to 100% activation probability for diode and Ho:YAG lasers. The stimulation threshold was lower for the Ho:YAG than the diode lasers across all examined spot sizes. The Ho:YAG also exhibited a nearly binary transition to 100% activation that can be mimicked with diode lasers by decreasing the pulse width. These results will help maximize the performance of laser diodes for INS.

Functional mapping of the connectivity of the brain is an essential procedure for neurosurgeons aiming for maximal resection of a brain tumor, while minimizing damage to the eloquent cortex. Being able to delineate the location of critical cortical areas in individual patients allows the surgeon to preserve sensorimotor and cognitive functions. Several methods exist for functional mapping, however, none are capable of delivering fast, label free mapping of the cortex with millimeter precision. In this work, we investigate the potential use of infrared neural stimulation (λ=1470nm) as a means of focal stimulation of the mouse cortex in vivo. Here, we show that the application of infrared neural stimulation induces in vivo cortical intracellular calcium signals in Layer II/III mouse neurons, using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f.