Region of interest determination algorithm of lensless calcium imaging datasets.

Advances in fluorescence imaging technology have been crucial to the progress of neuroscience. Whether it was specific expression of indicator proteins, detection of neurotransmitters, or miniaturization of fluorescence microscopes, fluorescence imaging has improved upon electrophysiology, the gold standard for monitoring brain activity, and enabled novel methods to sense activity in the brain. Hence, we developed a lightweight and compact implantable CMOS-based lensless Ca2+ imaging device for freely moving transgenic G-CaMP mouse experiments. However, without a lens system, determination of regions of interest (ROI) has proven challenging. Localization of fluorescence activity and separation of signal from noise are difficult. In this study, we report an ROI selection method using a series of adaptive binarizations with a gaussian method and morphological image processing. The parameters for each operation such as the kernel size, sigma and footprint size were optimized. We then validated the utility of the algorithm with simulated data and freely moving nociception experiments using the lensless devices. The device was implanted in the dorsal raphe nucleus to observe pain-related brain activity following a formalin test to stimulate pain. We observed significant increases in fluorescence activity after formalin injection compared to the control group when using the ROI determination algorithm.

Millimeter-Wave Band Electro-Optical Imaging System Using Polarization CMOS Image Sensor and Amplified Optical Local Oscillator Source.

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.

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.

Precise Temporal Control of Interferential Neural Stimulation via Phase Modulation.

OBJECTIVE: Noninvasive neural stimulation via temporally interferential (TI) electrical field is currently an area of interest as a noninvasive method of brain stimulation. The major limitation of TI stimulation is the difficulty of precise temporal control of the stimulation, due to the nature of the sinusoidal envelope generated by the interference. The purpose of this study was to investigate the possibility of improving interferential stimulation by introducing precise temporal control using phase modulation. METHODS: In conventional TI, a sinusoidal current is applied to two electrode pairs with slightly different frequencies, to cause interference. In this paper we describe phase modulation interference (PMI). Instead of shifting frequency, the phase of a sinusoidal wave was partially modulated, causing a transient increase or decrease of the envelope. The spatial distribution of envelope modulation amplitude by TI and PMI was visualized using both electromagnetic simulation and actual measurement using tissue phantom. RESULTS: The measured voltage transient in the tissue phantom produce a precise, temporally controlled pulse-like envelope using PMI. The spatial distributions of the amplitude of the envelope modulation by TI and PMI did not differ significantly, and were consistent with electromagnetic simulation. CONCLUSION: PMI allows precise temporal control of interferential stimulation, thus increasing the practical utility of interferential stimulation. SIGNIFICANCE: PMI improves interferential stimulation, allowing more temporally precise stimulation to neural tissue located distantly from the stimulating electrodes.

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.

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.

Safety assessment of semichronic suprachoroidal electrical stimulation to rabbit retina.

Confirming safety of chronic electrical stimulation is of prime importance for the practical use of visual prostheses. Here we applied electrical stimulation to eyes of freely-moving rabbits eight hour per day for one month. Examinations including fundus photo, optical coherence tomography (OCT), electrically evoked potentials (EEPs) were performed before and after one-month stimulation to detect tissue damage. No adverse effect caused by electrical stimulation was observed in electrophysiological and histological evaluation. We also found that there was no sign of morphological and electrochemical degradation of stimulating electrodes.

[Objective evaluation of visual fatigue for reading of radiographs displayed on medical-grade liquid-crystal displays].

The aim of this study was to evaluate visual fatigue objectively by measuring accommodation time and critical fusion frequency (CFF) before and after reading posteroanterior chest radiographs displayed on medical-grade liquid-crystal displays (LCDs) under different monitor conditions. A color LCD (500, 170 cd/m²) and a monochrome LCD (500 cd/m²) were used in this study. Six observers independently kept reading the radiographs for two hours to understand various lung nodules in the "Fatigue Session". Objective visual fatigue was measured by using the accommodation device and the CFF meter before and after the Fatigue Session. The ambient lighting of the laboratory was set at 35 lux. Both the accommodation time and the CFF between before and after the Fatigue Session indicated statistically significant differences (p<0.05). Our results on accommodation time and CFF before and after reading the radiographs on medical-grade LCDs indicated that visual fatigue could be evaluated objectively.

[Effect of ambient light variations on luminance ratio of CRT monitor and image contrast in ultrasonography].

Variation in the luminance ratio of a cathode ray tube(CRT)monitor and the ultrasonographic images at different levels of ambient light(0-150 lux)was investigated to obtain optimum ambient light in the ultrasonography suite. The maximum and minimum luminances of test patterns and ultrasonographic images were measured after three technicians independently optimized the brightness and contrast of the CRT monitor and ultrasonographic images at different levels of ambient light. Furthermore, the luminance ratio was calculated from the maximum luminance divided by the minimum luminance. When ambient light increased, it was difficult for the technicians to optimize the brightness and contrast settings of the CRT monitor to maintain a high luminance ratio at 0 lux. The luminance ratio decreased rapidly as ambient light increased up to 20 lux. However, the luminance ratio decreased gradually when ambient light was higher than 20 lux. It is necessary to take into consideration the ambient light to maintain a high luminance ratio of ultrasonographic images.

The development of a multichannel electrode array for retinal prostheses.

The development of a multielectrode array is the key issue for retinal prostheses. We developed a 10 x 10 platinum electrode array that consists of an 8-microm polyimide layer sandwiched between 5-microm polymonochloro-para-xylylene (parylene-C) layers. Each electrode was formed as a 30-microm-high bump by Pt/Au double-layer electroplating. We estimated the charge delivery capability (CDC) of the electrode by measuring the CDCs of two-channel electrode arrays. The dimensions of each electrode of the two-channel array were the same as those of each electrode formed on the 10 x 10 array. The results suggest that for cathodic-first (CF) pulses, 80% of electrodes surpassed our development target of 318 microC/cm2, which corresponds to the charge density of pulses of 500 micros duration and 200 microA amplitude for a 200-microm-diameter planar electrode.

Comparative sequencing of human and chimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence.

Despite their high degree of genomic similarity, reminiscent of their relatively recent separation from each other ( approximately 6 million years ago), the molecular basis of traits unique to humans vs. their closest relative, the chimpanzee, is largely unknown. This report describes a large-scale single-contig comparison between human and chimpanzee genomes via the sequence analysis of almost one-half of the immunologically critical MHC. This 1,750,601-bp stretch of DNA, which encompasses the entire class I along with the telomeric part of the MHC class III regions, corresponds to an orthologous 1,870,955 bp of the human HLA region. Sequence analysis confirms the existence of a high degree of sequence similarity between the two species. However, and importantly, this 98.6% sequence identity drops to only 86.7% taking into account the multiple insertions/deletions (indels) dispersed throughout the region. This is functionally exemplified by a large deletion of 95 kb between the virtual locations of human MICA and MICB genes, which results in a single hybrid chimpanzee MIC gene, in a segment of the MHC genetically linked to species-specific handling of several viral infections (HIV/SIV, hepatitis B and C) as well as susceptibility to various autoimmune diseases. Finally, if generalized, these data suggest that evolution may have used the mechanistically more drastic indels instead of the more subtle single-nucleotide substitutions for shaping the recently emerged primate species.