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In this study, we developed and demonstrated a millimeter-wave electric field imaging system using an electro-optic crystal and a highly sensitive polarization measurement technique using a polarization image sensor, which was fabricated using a 0.35-µm standard CMOS process. The polarization image sensor was equipped with differential amplifiers that amplified the difference between the 0° and 90° pixels. With the amplifier, the signal-to-noise ratio at low incident light levels was improved. Also, an optical modulator and a semiconductor optical amplifier were used to generate an optical local oscillator (LO) signal with a high modulation accuracy and sufficient optical intensity. By combining the amplified LO signal and a highly sensitive polarization imaging system, we successfully performed millimeter-wave electric field imaging with a spatial resolution of 30×60 µm at a rate of 1 FPS, corresponding to 2400 pixels/s.
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Objective: Current neuronal imaging methods mostly use bulky lenses that either impede animal behavior or prohibit multi-depth imaging. To overcome these limitations, we developed a lightweight lensless biophotonic system for neuronal imaging, enabling compact and simultaneous visualization of multiple brain layers.Approach: Our developed 'CIS-NAIST' device integrates a micro-CMOS image sensor, thin-film fluorescence filter, micro-LEDs, and a needle-shaped flexible printed circuit. With this device, we monitored neuronal calcium dynamics during seizures across the different layers of the hippocampus and employed machine learning techniques for seizure classification and prediction.Main results: The CIS-NAIST device revealed distinct calcium activity patterns across the CA1, molecular interlayer, and dentate gyrus. Our findings indicated an elevated calcium amplitude activity specifically in the dentate gyrus compared to other layers. Then, leveraging the multi-layer data obtained from the device, we successfully classified seizure calcium activity and predicted seizure behavior using Long Short-Term Memory and Hidden Markov models.Significance: Taken together, our 'CIS-NAIST' device offers an effective and minimally invasive method of seizure monitoring that can help elucidate the mechanisms of temporal lobe epilepsy.
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Cálcio , Hipocampo , Convulsões , Animais , Hipocampo/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Cálcio/metabolismo , Masculino , Agulhas , Ratos , SemicondutoresRESUMO
We propose and demonstrate a method for equivalent time sampling using image sensors to selectively detect only the target frequency. Shortening the exposure time of the image sensor and using equivalent time sampling allows for the detection of frequency components that are higher than the frame rate. However, the imaging system in our previous work was also sensitive to the frequency component at 1/4 of the frame rate. In this study, we control the phase relationship between the exposure time and observed signal by inserting an additional interval once every four frames to detect the target frequency selectively. With this technique, we conducted electric field imaging based on the electro-optic effect under high noise conditions in the low-frequency band to which the conventional method is sensitive. The results demonstrated that the proposed method improved the signal-to-noise ratio.
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Goreisan is a Kampo medicine used to treat headaches associated with climate change. Here, by using an implantable complementary metal-oxide-semiconductor (CMOS) device, we evaluated the effects of Goreisan and loxoprofen on cerebral blood flow (CBF) dynamics associated with barometric pressure fluctuations in freely moving mice. In the vehicle group, decreasing barometric pressure increased CBF that was prevented by Goreisan and loxoprofen. Notably, Goreisan, but not loxoprofen, reduced CBF after returning to atmospheric pressure. These results indicate that, unlike the mechanism of action of antipyretic analgesics, Goreisan normalizes CBF abnormalities associated with barometric pressure fluctuations by actively reducing CBF increase.
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Pressão Atmosférica , Circulação Cerebrovascular , Medicamentos de Ervas Chinesas , Fenilpropionatos , Feminino , Animais , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Hybrid emission filters, comprising an interference filter and an absorption filter, exhibit high excitation light rejection performance and can act as lensless fluorescent devices. However, it has been challenging to produce them in large batches over a large area. In this study, we propose and demonstrate a method for transferring a Si substrate, on which the hybrid filter is deposited, onto an image sensor by attaching it to the sensor and removing the substrate via plasma etching. Through this method, we can transfer uniform filters onto fine micrometer-sized needle devices and millimeter-sized multisensor chips. Optical evaluation reveals that the hybrid filter emits light in the 500 to 560 nm range, close to the emission region of green fluorescent protein (GFP). Furthermore, by observing the fluorescence emission from the microbeads, a spatial resolution of 12.11 µm is calculated. In vitro experiments confirm that the fabricated device is able to discriminate GFP emission patterns from brain slices.
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In this research, we combined our ultralight micro-imaging device for calcium imaging with microdialysis to simultaneously visualize neural activity in the dorsal raphe nucleus (DRN) and measure serotonin release in the central nucleus of the amygdala (CeA) and the anterior cingulate cortex (ACC). Using this platform, we observed brain activity following nociception induced by formalin injection in the mouse's hind paw. Our device showed that DRN fluorescence intensity increased after formalin injection, and the increase was highly correlated with the elevation in serotonin release in both the CeA and ACC. The increase in calcium fluorescence intensity occurred during the acute and inflammatory phases, which suggests the biphasic response of nociceptive pain. Furthermore, we found that the increase in fluorescence intensity was positively correlated with mouse licking behavior. Lastly, we compared the laterality of pain stimulation and found that DRN fluorescence activity was higher for contralateral stimulation. Microdialysis showed that CeA serotonin concentration increased only after contralateral stimulation, while ACC serotonin release responded bilaterally. In conclusion, our study not only revealed the inter-regional serotonergic connection among the DRN, the CeA, and the ACC, but also demonstrated that our device is feasible for multi-site implantation in conjunction with a microdialysis system, allowing the simultaneous multi-modal observation of different regions in the brain.
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Dor Nociceptiva , Serotonina , Camundongos , Animais , Serotonina/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Microdiálise , Cálcio , Sinalização do CálcioRESUMO
A readout device for a dual-functional neural observation system is presented. The authors separately developed the reading operation of an implantable CMOS image sensor and a setup for fast-scan cyclic voltammetry and implemented them together in a microcontroller-based device. The developed imaging readout device with a size of [Formula: see text] can reach the highest reading rate of 160 fps with a 120×268 pixel image sensor. The voltammetry function was verified through an experiment using commercial carbon fiber electrodes in phosphate-buffered saline. When the imaging is sequentially operated with 400 V/s-scan rate voltammetry from -0.4 to 1.3 V, the system can operate at up to 60 fps. With this system, calcium imaging and dopamine recording in a freely behaving mouse can be achieved together in a simpler manner. This study aims to be the basis for the development of an implantable multi-functional sensor.
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Cálcio , Imagem Óptica , Animais , Fibra de Carbono , Dopamina , Camundongos , CintilografiaRESUMO
SIGNIFICANCE: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings. AIM: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions. APPROACH: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λcenter: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions. RESULTS: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed. CONCLUSIONS: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.
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Encéfalo , Diagnóstico por Imagem , Animais , Encéfalo/fisiologia , Hemodinâmica , Camundongos , Córtex Somatossensorial/diagnóstico por imagemRESUMO
Dopamine (DA) is the key regulator of reward behavior. The DA neurons in the ventral tegmental area (VTA) and their projection areas, which include the prefrontal cortex (PFC), nucleus accumbens (NAc), and amygdala, play a primary role in the process of reward-driven behavior induced by the drugs of addiction, including nicotine and alcohol. In our previous study, we developed a novel platform consisting of micro-LED array devices to stimulate a large area of the brain of rats and monkeys with photo-stimulation and a microdialysis probe to estimate the DA release in the PFC. Our results suggested that the platform was able to detect the increased level of dopamine in the PFC in response to the photo-stimulation of both the PFC and VTA. In this study, we used this platform to photo-stimulate the VTA neurons in both ChrimsonR-expressing (non-specific) wild and dopamine transporter (DAT)-Cre (dopamine specific) mice, and measured the dopamine release in the nucleus accumbens shell (NAcShell). We measured the DA release in the NAcShell in response to optogenetic stimulation of the VTA neurons and investigated the effect of GABAergic neurons on dopaminergic neurons by histochemical studies. Comparing the photo-stimulation frequency of 2 Hz with that of 20 Hz, the change in DA concentration at the NAcShell was greater at 20 Hz in both cases. When ChrimsonR was expressed specifically for DA, the release of DA at the NAcShell increased in response to photo-stimulation of the VTA. In contrast, when ChrimsonR was expressed non-specifically, the amount of DA released was almost unchanged upon photo-stimulation. However, for nonspecifically expressed ChrimsonR, intraperitoneal injection of bicuculline, a competitive antagonist at the GABA-binding site of the GABAA receptor, also significantly increased the release of DA at the NAcShell in response to photo-stimulation of the VTA. The results of immunochemical staining confirm that GABAergic neurons in the VTA suppress DA activation, and also indicate that alterations in GABAergic neurons may have serious downstream effects on DA activity, NAcShell release, and neural adaptation of the VTA. This study also confirms that optogenetics technology is crucial to study the relationship between the mesolimbic dopaminergic and GABAergic neurons in a neural-specific manner.
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Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Neurônios Dopaminérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Optogenética/métodos , Área Tegmentar Ventral/metabolismo , Animais , Bicuculina/farmacologia , Channelrhodopsins/genética , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Camundongos , Núcleo Accumbens/metabolismo , Imagem ÓpticaRESUMO
SIGNIFICANCE: Gene expression analysis is an important fundamental area of biomedical research. However, live gene expression imaging has proven challenging due to constraints in conventional optical devices and fluorescent reporters. AIM: Our aim is to develop smaller, more cost-effective, and versatile imaging capabilities compared with conventional devices. Bioluminescence reporter-based gene expression analysis was targeted due to its advantages over fluorescence-based imaging. APPROACH: We created a small compact imaging system using micro-CMOS image sensors (µCIS). The µCIS model had an improved pixel design and a patterned absorption filter array to detect the low light intensity of bioluminescence. RESULTS: The device demonstrated lower dark current, lower temporal noise, and higher sensitivity compared with previous designs. The filter array enabled us to subtract dark current drift and attain a clearer light signal. These improvements allowed us to measure bioluminescence reporter-based gene expression in living mammalian cells. CONCLUSION: Using our µCIS system for bioluminescence imaging in the future, the device can be implanted in vivo for simultaneous gene expression imaging, behavioral analysis, and optogenetic modulation.
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Medições Luminescentes , Animais , Expressão Gênica , Genes ReporterRESUMO
In this study, we propose a complementary-metal-oxide-semiconductor (CMOS) image sensor with a self-resetting system demonstrating a high signal-to-noise ratio (SNR) to detect small intrinsic signals such as a hemodynamic reaction or neural activity in a mouse brain. The photodiode structure was modified from N-well/P-sub to P+/N-well/P-sub to increase the photodiode capacitance to reduce the number of self-resets required to decrease the unstable stage. Moreover, our new relay board was used for the first time. As a result, an effective SNR of over 70 dB was achieved within the same pixel size and fill factor. The unstable state was drastically reduced. Thus, we will be able to detect neural activity. With its compact size, this device has significant potential to become an intrinsic signal detector in freely moving animals. We also demonstrated in vivo imaging with image processing by removing additional noise from the self-reset operation.
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Fluorescence imaging devices have been indispensable in elucidating the workings of the brain in living animals, including unrestrained, active ones. Various devices are available, each with their own strengths and weaknesses in terms of many factors. We have developed CMOS-based needle-type imaging devices that are small and lightweight enough to be doubly implanted in freely moving mice. The design also allowed angled implantations to avoid critical areas. We demonstrated the utility of the devices by using them on GCaMP6 mice in a formalin test experiment. Simultaneous implantations to the capsular-lateral central amygdala (CeLC) and dorsal raphe nucleus (DRN) were proven to be safe and did not hinder the execution of the study. Analysis of the collected calcium signaling data, supported by behavior data, showed increased activity in both regions as a result of pain stimulation. Thus, we have successfully demonstrated the various advantages of the device in its application in the pain experiment.
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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.
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Optogenética , Semicondutores , Estimulação LuminosaRESUMO
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.
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Adenoviridae/fisiologia , Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Optogenética , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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Propranolol, a ß-adrenergic receptor blocker, is one of the most commonly used prophylactic drugs for migraines. Cortical spreading depression (CSD) is the propagation wave of neuronal excitation along with cerebral blood flow (CBF) changes over the cerebral cortex and has been implicated in the pathological process of migraine auras and its pain response. However, the effect of propranolol on CSD-related CBF changes and behavioral responses remains poorly understood. In this study, we measured CSD-related CBF responses using a micro-device with a green light emitting diode (LED) and micro-complementary-metal-oxide-semiconductor (CMOS) image sensor and evaluated pain-related reduced locomotor activity in mice. An injection of KCl into the visual cortex led to CSD-related CBF changes; however, propranolol prevented the increase in CBF as well as delayed the propagation velocity in KCl-induced CSD. Furthermore, an injection of KCl reduced locomotor activity and induced freezing behavior in awake and freely moving mice, which were prevented by propranolol treatment. These results suggest that the modulation of CSD-related CBF responses by the blockade of ß-adrenergic receptor contributes to its prophylactic effects on migraines.
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Circulação Cerebrovascular/efeitos dos fármacos , Transtornos de Enxaqueca/prevenção & controle , Propranolol/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Enxaqueca/diagnóstico por imagem , Transtornos de Enxaqueca/fisiopatologia , Atividade Motora/efeitos dos fármacos , Dor/tratamento farmacológico , Dor/fisiopatologia , Cloreto de Potássio/administração & dosagemRESUMO
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.
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Ensaio de Imunoadsorção Enzimática/métodos , Medições Luminescentes/métodos , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática/instrumentação , Humanos , Microfluídica , Fotodegradação , Transistores EletrônicosRESUMO
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.
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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.
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Encéfalo/citologia , Encéfalo/fisiologia , Cálcio/metabolismo , Comunicação Celular , Imagem Molecular , Imagem Óptica , Optogenética , Próteses e Implantes , Animais , Técnicas de Cultura de Células , Linhagem Celular , Camundongos , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Optogenética/instrumentação , Optogenética/métodos , Estimulação LuminosaRESUMO
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.