CMOS-Based Neural Interface Device for Optogenetics.

Optical and electronic neural interface devices based on CMOS technology are presented. Concept, design strategy, and fabrication of the CMOS-based optoelectronic neural interface devices are described. The devices are based on a technology of implantable CMOS image sensor. To realize addressable local optical stimulation, blue light-emitting diode array chip was integrated on the implantable CMOS image sensors. Functional demonstrations of the devices are also presented. Optical stimulation capability was demonstrated in both in vitro and in vivo experiments. Further perspective including wireless device architecture is also presented.

Catalytic asymmetric synthesis of 3,3'-diaryloxindoles as triarylmethanes with a chiral all-carbon quaternary center: phase-transfer-catalyzed S(N)Ar reaction.

Catalytic asymmetric synthesis of unsymmetrical triarylmethanes with a chiral all-carbon quaternary center was achieved by using a chiral bifunctional quaternary phosphonium bromide catalyst in the S(N)Ar reaction of 3-aryloxindoles under phase-transfer conditions. The presence of a urea moiety in the chiral phase-transfer catalyst was important for obtaining high enantioselectivity in this reaction.

Balancing risk-return decisions by manipulating the mesofrontal circuits in primates.

Decision-making is always coupled with some level of risk, with more pathological forms of risk-taking decisions manifesting as gambling disorders. In macaque monkeys trained in a high risk-high return (HH) versus low risk-low return (LL) choice task, we found that the reversible pharmacological inactivation of ventral Brodmann area 6 (area 6V) impaired the risk dependency of decision-making. Selective optogenetic activation of the mesofrontal pathway from the ventral tegmental area (VTA) to the ventral aspect of 6V resulted in stronger preference for HH, whereas activation of the pathway from the VTA to the dorsal aspect of 6V led to LL preference. Finally, computational decoding captured the modulations of behavioral preference. Our results suggest that VTA inputs to area 6V determine the decision balance between HH and LL.

Image sensor pixel with on-chip high extinction ratio polarizer based on 65-nm standard CMOS technology.

In this study, we demonstrate a polarization sensitive pixel for a complementary metal-oxide-semiconductor (CMOS) image sensor based on 65-nm standard CMOS technology. Using such a deep-submicron CMOS technology, it is possible to design fine metal patterns smaller than the wavelengths of visible light by using a metal wire layer. We designed and fabricated a metal wire grid polarizer on a 20 × 20 µm(2) pixel for image sensor. An extinction ratio of 19.7 dB was observed at a wavelength 750 nm.

CMOS image sensor integrated with micro-LED and multielectrode arrays for the patterned photostimulation and multichannel recording of neuronal tissue.

We developed a complementary metal oxide semiconductor (CMOS) integrated device for optogenetic applications. This device can interface via neuronal tissue with three functional modalities: imaging, optical stimulation and electrical recording. The CMOS image sensor was fabricated on 0.35 µm standard CMOS process with built-in control circuits for an on-chip blue light-emitting diode (LED) array. The effective imaging area was 2.0 × 1.8 mm². The pixel array was composed of 7.5 × 7.5 µm² 3-transistor active pixel sensors (APSs). The LED array had 10 × 8 micro-LEDs measuring 192 × 225 µm². We integrated the device with a commercial multichannel recording system to make electrical recordings.

Optogenetic induction of hibernation-like state with modified human Opsin4 in mice.

We recently determined that the excitatory manipulation of Qrfp-expressing neurons in the preoptic area of the hypothalamus (quiescence-inducing neurons [Q neurons]) induced a hibernation-like hypothermic/hypometabolic state (QIH) in mice. To control the QIH with a higher time resolution, we develop an optogenetic method using modified human opsin4 (OPN4; also known as melanopsin), a G protein-coupled-receptor-type blue-light photoreceptor. C-terminally truncated OPN4 (OPN4dC) stably and reproducibly induces QIH for at least 24 h by illumination with low-power light (3 µW, 473 nm laser) with high temporal resolution. The high sensitivity of OPN4dC allows us to transcranially stimulate Q neurons with blue-light-emitting diodes and non-invasively induce the QIH. OPN4dC-mediated QIH recapitulates the kinetics of the physiological changes observed in natural hibernation, revealing that Q neurons concurrently contribute to thermoregulation and cardiovascular function. This optogenetic method may facilitate identification of the neural mechanisms underlying long-term dormancy states such as sleep, daily torpor, and hibernation.

Photoactivatable oncolytic adenovirus for optogenetic cancer therapy.

Virotherapy using oncolytic adenovirus is an effective anticancer strategy. However, the tumor selectivity of oncolytic adenoviruses is not enough high. To develop oncolytic adenovirus with a low risk of off-tumor toxicity, we constructed a photoactivatable oncolytic adenovirus (paOAd). In response to blue light irradiation, the expression of adenoviral E1 genes, which are necessary for adenoviral replication, is induced and replication of this adenovirus occurs. In vitro, efficient lysis of various human cancer cell lines was observed by paOAd infection followed by blue light irradiation. Importantly, there was no off-tumor toxicity unless the cells were irradiated by blue light. In vivo, tumor growth in a subcutaneous tumor model and a mouse model of liver cancer was significantly inhibited by paOAd infection followed by blue light irradiation. In addition, paOAd also showed a therapeutic effect on cancer stem cells. These results suggest that paOAd is useful as a safe and therapeutically effective cancer therapy.

Improved Charge Pump Design and Ex Vivo Experimental Validation of CMOS 256-Pixel Photovoltaic-Powered Subretinal Prosthetic Chip.

An improved design of CMOS 256-pixel photovoltaic-powered implantable chip for subretinal prostheses is presented. In the proposed subretinal chip, a high-efficiency fully-integrated 4× charge pump is designed and integrated with on-chip photovoltaic (PV) cells and a 256-pixel array with active pixel sensors (APS) for image light sensing, biphasic constant current stimulators, and electrodes. Thus the PV voltage generated by infrared (IR) light can be boosted to above 1V so that the charge injection is increased. The proposed chip adopts the 32-phase divisional power supply scheme (DPSS) to reduce the required supply current and thus the required area of the PV cells. The proposed chip is designed and fabricated in 180-nm CMOS image sensor (CIS) technology and post-processed with biocompatible IrOx electrodes and silicone packaging. From the electrical measurement results, the measured stimulation frequency is 28.3 Hz under the equivalent electrode impedance load. The measured maximum output stimulation current is 7.1 µA and the amount of injected charges per pixel is 7.36 nC under image light intensity of 3200 lux and IR light intensity of 100 mW/cm2. The function of the proposed chip has been further validated successfully with the ex vivo experimental results by recording the electrophysiological responses of retinal ganglion cells (RGCs) of retinas from retinal degeneration (rd1) mice with a multi-electrode array (MEA). The measured average threshold injected charge is about 3.97 nC which is consistent with that obtained from the patch clamp recording on retinas from wild type (C57BL/6) mice with a single electrode pair.

Implantable CMOS Biomedical Devices.

The results of recent research on our implantable CMOS biomedical devices are reviewed. Topics include retinal prosthesis devices and deep-brain implantation devices for small animals. Fundamental device structures and characteristics as well as in vivo experiments are presented.

Chronic brain blood-flow imaging device for a behavioral experiment using mice.

A chronic brain blood-flow imaging device was developed for cerebrovascular disease treatment. This device comprises a small complementary metal-oxide semiconductor image sensor and a chronic fiber-optic plate window on a mouse head. A long-term cerebral blood-flow imaging technique was established in a freely moving mouse. Brain surface images were visible for one month using the chronic FOP window. This device obtained brain surface images and blood-flow velocity. The blood-flow changes were measured in behavioral experiments using this device. The chronic brain blood-flow imaging device may contribute to determining the cause of cerebrovascular disease and the development of cerebrovascular disease treatment.

One-chip sensing device (biomedical photonic LSI) enabled to assess hippocampal steep and gradual up-regulated proteolytic activities.

We developed an implantable one-chip biofluoroimaging device (termed biomedical photonic LSI; BpLSI) which enabled real-time molecular imaging with conventional electrophysiology in vivo in deep brain areas. The multimodal LSI enabled long-term sequential imaging of the fluorescence emitted by proteolysis-linked fluorogenic substrate. Using the BpLSI, we observed a process of stimulation-dependent modulation at synapse with multi-site (16 x 19 pixel) in widespread area and a high-speed video rate, and found that the gradual up-regulated proteolytic activity in a wide range of hippocampal CA1 area and the steep activity in local area, indicating that the proteolysis system is a basis for the fixation of long-term potentiation in post-excited synapses in the hippocampus. Mathematical data analysis confirmed the direct involvement of functional proteolysis for neural plasticity.

Implantable Microimagers.

Implantable devices such as cardiac pacemakers, drug-delivery systems, and defibrillators have had a tremendous impact on the quality of live for many disabled people. To date, many devices have been developed for implantation into various parts of the human body. In this paper, we focus on devices implanted in the head. In particular, we describe the technologies necessary to create implantable microimagers. Design, fabrication, and implementation issues are discussed vis-à-vis two examples of implantable microimagers; the retinal prosthesis and in vivo neuro-microimager. Testing of these devices in animals verify the use of the microimagers in the implanted state. We believe that further advancement of these devices will lead to the development of a new method for medical and scientific applications.

Highly sensitive lens-free fluorescence imaging device enabled by a complementary combination of interference and absorption filters.

We report a lens-free fluorescence imaging device using a composite filter composed of an interference filter and an absorption filter, each applied to one side of a fiber optic plate (FOP). The transmission of angled excitation light through the interference filter is absorbed by the absorption filter. The auto-fluorescence of the absorption filter is reduced by the reflection from the interference filter of normally incident excitation light. As a result, high-performance rejection of excitation light is achieved in a lens-free device. The FOP provides a flat, hard imaging device surface that does not degrade the spatial resolution. We demonstrate excitation rejection of approximately 108:1 at a wavelength of 450 nm in a fabricated lens-free device. The resolution of fluorescence imaging is approximately 12 µm. Time-lapse imaging of cells containing green fluorescent protein was performed in a 5-µm thin-film chamber. The small dimensions of the device allow observation of cell culturing in a CO2 incubator. We also demonstrate that the proposed lens-free filter is compatible with super-resolution bright-field imaging techniques. These features open a way to develop a high-performance, dual-mode, lens-free imaging device that is expected to be a powerful tool for many applications, such as imaging of labeled cells and point-of-care assay.

Compact Lensless Fluorescence Counting System for Single Molecular Assay.

In digital enzyme-linked immunosorbent assay, which is used for biomarker detection and diagnosis, the concentration of target biomarkers is estimated by counting the number of fluorescence chambers in the microchamber array. We propose a compact system for counting fluorescent chambers. Our system consists of three components: a micro-reaction chamber array, an absorption filter for attenuating excitation light, and a photodetector. The absorption filter has a micro-light-pipe array (m-LPA) structure. A stacked photodiode CMOS image sensor (CIS), which can discriminate color, is applied as a photodetector. This paper describes the fabrication process enabling thin m-LPA chips. The unique low-noise characteristics of the stacked photodiode CIS that attains high sensitivity by adopting the 4T-APS configuration are explained. Furthermore, a detection method using the photobleaching phenomenon is proposed for high-sensitivity fluorescence detection. This method suggests that fluorescence by a single molecular enzyme can be detected within 30 min of the start of the fluorescence reaction.

Real time in vivo imaging and measurement of serine protease activity in the mouse hippocampus using a dedicated complementary metal-oxide semiconductor imaging device.

The aim of the present study is to demonstrate the application of complementary metal-oxide semiconductor (CMOS) imaging technology for studying the mouse brain. By using a dedicated CMOS image sensor, we have successfully imaged and measured brain serine protease activity in vivo, in real-time, and for an extended period of time. We have developed a biofluorescence imaging device by packaging the CMOS image sensor which enabled on-chip imaging configuration. In this configuration, no optics are required whereby an excitation filter is applied onto the sensor to replace the filter cube block found in conventional fluorescence microscopes. The fully packaged device measures 350 microm thick x 2.7 mm wide, consists of an array of 176 x 144 pixels, and is small enough for measurement inside a single hemisphere of the mouse brain, while still providing sufficient imaging resolution. In the experiment, intraperitoneally injected kainic acid induced upregulation of serine protease activity in the brain. These events were captured in real time by imaging and measuring the fluorescence from a fluorogenic substrate that detected this activity. The entire device, which weighs less than 1% of the body weight of the mouse, holds promise for studying freely moving animals.

Functional verification of pulse frequency modulation-based image sensor for retinal prosthesis by in vitro electrophysiological experiments using frog retina.

The functioning of a 16 x 16 pixel pulse frequency modulation (PFM) image sensor for retinal prosthesis is verified through in vitro electrophysiological experiments using detached frog retinas. This image sensor is a prototype for demonstrating the application to in vitro electrophysiological experiments. Each pixel of the image sensor consists of a pulse generator (PFM photosensor), a stimulus circuit, and a stimulus electrode (Al bonding pad). The image sensor is fabricated using standard 0.6 microm CMOS technology. For in vitro electrophysiological experiments, a Pt/Au stacked electrode is formed on the Al bonding pad of each pixel and the entire sensor is fixed in epoxy resin. The PFM image sensor is confirmed experimentally to provide electrical stimulus to the retinal cells in a detached frog retina.

CMOS-based opto-electronic neural interface devices for optogenetics.

CMOS-based opto-electronic neural interface devices are presented. The devices are designed with target application of in vitro and in vivo optogenetics. Two types of the opto-electronic neural interface devices are presented. One is single-chip type device for on-chip optogenetics, and the other is multi-chip type device with flexibility and wide-area coverage for in vivo optogenetics on brain. Design, packaging and functional evaluations are presented.

"Optical communication with brain cells by means of an implanted duplex micro-device with optogenetics and Ca(2+) fluoroimaging".

To better understand the brain function based on neural activity, a minimally invasive analysis technology in a freely moving animal is necessary. Such technology would provide new knowledge in neuroscience and contribute to regenerative medical techniques and prosthetics care. An application that combines optogenetics for voluntarily stimulating nerves, imaging to visualize neural activity, and a wearable micro-instrument for implantation into the brain could meet the abovementioned demand. To this end, a micro-device that can be applied to the brain less invasively and a system for controlling the device has been newly developed in this study. Since the novel implantable device has dual LEDs and a CMOS image sensor, photostimulation and fluorescence imaging can be performed simultaneously. The device enables bidirectional communication with the brain by means of light. In the present study, the device was evaluated in an in vitro experiment using a new on-chip 3D neuroculture with an extracellular matrix gel and an in vivo experiment involving regenerative medical transplantation and gene delivery to the brain by using both photosensitive channel and fluorescent Ca(2+) indicator. The device succeeded in activating cells locally by selective photostimulation, and the physiological Ca(2+) dynamics of neural cells were visualized simultaneously by fluorescence imaging.

Fabrication and functional demonstration of a smart electrode with a built-in CMOS microchip for neural stimulation of a retinal prosthesis.

In this study, we propose an advanced architecture of a smart electrode for neural stimulation of a retinal prosthesis. A feature of the proposed architecture is embedding CMOS microchips into the core of the stimulus electrodes. Microchip integration without dead space on the array is possible. Additionally, higher durability can be expected because the microchips are protected by the stimulus electrodes like a metal casing. Dedicated circular-shaped CMOS microchips were designed and fabricated. The microchip measured 400 µm in diameter. Stimulus electrodes that had a microcavity for embedding the microchip were also fabricated. In the assembly process, the CMOS microchip was mounted on a flexible substrate, and then the stimulus electrode was mounted to cover the microchip. The microchip was completely built into the inside of the electrode. By performing an ex-vivo experiment using the extracted eyeball of a pig, stimulus function of the electrode was demonstrated successfully.