Exposure Time Control Method for Higher Intermediate Frequency in Optical Heterodyne Imaging and Its Application to Electric-Field Imaging Based on Electro-Optic Effect.

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.

Thin and Scalable Hybrid Emission Filter via Plasma Etching for Low-Invasive Fluorescence Detection.

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.

Multi-Region Microdialysis Imaging Platform Revealed Dorsal Raphe Nucleus Calcium Signaling and Serotonin Dynamics during Nociceptive Pain.

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.

Development of Compact Readout Device for Neural Observation System using Fluorescence Imaging and Fast-scan Cyclic Voltammetry.

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.

Modular head-mounted cortical imaging device for chronic monitoring of intrinsic signals in mice.

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.

Investigating the Influence of GABA Neurons on Dopamine Neurons in the Ventral Tegmental Area Using Optogenetic Techniques.

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.

Ultrasmall compact CMOS imaging system for bioluminescence reporter-based live gene expression analysis.

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.

Self-Reset Image Sensor With a Signal-to-Noise Ratio Over 70 dB and Its Application to Brain Surface Imaging.

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.

Simultaneous CMOS-Based Imaging of Calcium Signaling of the Central Amygdala and the Dorsal Raphe Nucleus During Nociception in Freely Moving Mice.

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.

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.

Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue.

The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 µm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation.