Hiroaki Takehara

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.

An Implantable CMOS Image Sensor With Self-Reset Pixels for Functional Brain Imaging

In this paper, we propose and demonstrate an implantable CMOS image sensor with self-resetting pixels. The self-resetting function is implemented using a four-transistor Schmitt trigger inverter. The pixel has no counter for the number of self-resets, because the application does not require radiometric (linear) response. The pixel is fabricated using the 0.35-μm 2-poly 4-metal standard CMOS technology, which results in the pixel size of 15 μm×15 μm and a fill factor of 31%. The effective peak signal-to-noise ratio is >59 dB. An image sensor prototype comprising a 60 × 134 pixel array is designed, and an implantable device is fabricated. As an example imaging experiment, we demonstrate blood-flow imaging of a rat-brain surface using the sensor. Intensity-change images are successfully obtained from the self-resetting pixel outputs with the image processing.

Implantable imaging device for brain functional imaging system using flavoprotein fluorescence

The autofluorescence of mitochondrial flavoprotein is very useful for functional brain imaging because the fluorescence intensity of flavoprotein changes as per neural activities. In this study, we developed an implantable imaging device for green fluorescence imaging and detected fluorescence changes of flavoprotein associated with visual stimulation using the device. We examined the device performance using anesthetized mice. We set the device on the visual cortex and measured fluorescence changes of flavoprotein in response to visual stimulation. A full-field sinusoidal grating with a vertical orientation was used for applying to activate the visual cortex. We successfully observed visually evoked fluorescence changes in the mouse visual cortex using our implantable device. This result suggests that we can observe the fluorescence changes of flavoprotein associated with visual stimulation in a freely moving mouse by using this technology.

An implantable green fluorescence imaging device using absorption filters with high excitation light rejection ratio

We have developed an implantable complementary-metal-oxide-semiconductor (CMOS) imaging device for green fluorescence imaging to observe various neural activities of the mouse brain in a freely moving state. The device comprises a CMOS image sensor, blue LEDs as excitation light sources, and absorption filters to enable real-time green fluorescence imaging. To observe weak green fluorescence reactions such as that of green fluorescent protein (GFP), we achieved efficient excitation light rejection using a combination of dedicated absorption filters and achieved the detection of GFP positive cells from mouse brain slices. It is expected that high-sensitivity green fluorescence imaging of neural activities in a freely moving mouse will be possible using this technology.

Implantable Microimaging Device for Observing Brain Activities of Rodents

In this review, we present an implantable microimaging device to observe brain activities of small experimental animals such as mice and rats. Three categories of such devices are described: an optical fiber system, a head-mountable fluorescent microscope, and an ultrasmall image sensor that can be directly implanted into the brain. Among them, we focus on the third one, because this is a powerful tool to explore brain activities in deep brain region in a freely moving mouse. The device structure and performance are shown with some examples of deep brain images of mice.

Micro-light-pipe array with an excitation attenuation filter for lensless digital enzyme-linked immunosorbent assay

Digital enzyme-linked immunosorbent assay (ELISA) is used for detecting various biomarkers with hypersensitivity. We have been developing compact systems by replacing the fluorescence microscope with a CMOS image sensor. Here, we propose a micro-light-pipe array structure made of metal filled with dye-doped resin, which can be used as a fabrication substrate of the micro-reaction-chamber array of digital ELISA. The possibility that this structure enhances the coupling efficiency for fluorescence was simulated using a simple model. To realize the structure, we fabricated a 30-µm-thick micropipe array by copper electroplating around a thick photoresist pattern. The typical diameter of each fabricated micropipe was 10 µm. The pipes were filled with yellow-dye-doped epoxy resin. The transmittance ratio of fluorescence and excitation light could be controlled by adjusting the doping concentration. We confirmed that an angled excitation light incidence suppressed the leakage of excitation light.

Implantable micro-optical semiconductor devices for optical theranostics in deep tissue

Optical therapy and diagnostics using photoactivatable molecular tools are promising approaches in medical applications; however, a method for the delivery of light deep inside biological tissues remains a challenge. Here, we present a method of illumination and detection of light using implantable micro-optical semiconductor devices. Unlike in conventional transdermal light delivery methods using low-energy light (>620 nm or near-infrared light), in our method, high-energy light (470 nm) can also be used for illumination. Implanted submillimeter-sized light-emitting diodes were found to provide sufficient illumination (0.6–4.1 mW/cm2), and a complementary metal–oxide–semiconductor image sensor enabled the detection of fluorescence signals.

Performance Improvement of a Micro-stimulus Electrode for Retinal Prosthesis by Introducing a High-Performance Material and a Three-Dimensional Structure

High-performance electrodes intended for retinal prosthesis were fabricated and evaluated. Iridium oxide (IrOx) was introduced as a high-performance material. A three-dimensional (3D) structure was also introduced to enlarge the electrode’s surface area. We tried to improve the electrode performance by combining these approaches, even if the electrode was miniaturized. The effectiveness of IrOx was demonstrated through electrochemical evaluation by comparing it with Pt. IrOx showed 1.6–6 times higher performance for the injection of stimulus pulse current than Pt. The performance of the 3D electrode compared with a planar electrode was also evaluated. Accordingly, the 3D electrode showed 2–4 times higher performance than the planar electrode by surface area enlargement. An ex vivo validation of the stimulus performance was conducted to demonstrate its practical use. A fabricated electrode was implanted in an extracted pig eyeball and the electrochemical performance was evaluated. The fabricated electrode showed sufficient performance of the retinal stimulation, with a high margin of safety. The proposed approach was successfully demonstrated as a stimulus electrode candidate for use in next-generation retinal prosthesis.

In Vitro Long-Term Performance Evaluation and Improvement in the Response Time of CMOS-Based Implantable Glucose Sensors

This article confirmed that the sensor retains its measurement capability for more than 150 days in a saline solution. In addition, an optimization of the sensor structure for obtaining an improved response time was carried out. A response time as short as that of the conventional self-monitoring of blood glucose (SMBG) technology was obtained with the optimized sensor structure.

High coupling efficiency contact imaging system having micro light pipe array for a digital enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) is a diagnostic technique used for detecting the presence of viruses or tumor markers. To detect target biomarkers, antigen-antibody reactions followed by fluorescent reactions are carried out in an ELISA. Recently, digital ELISAs have been proposed to achieve higher sensitivity. In the digital ELISA, fluorescent reactions are carried out in an array of femtoliter-scale microchambers. The concentration of the target biomarkers is determined by counting the number of microchambers with and without fluorescence using a fluorescence microscope. We have been developing compact digital ELISA systems by replacing the fluorescence microscope with a dedicated stacked photodiode CMOS image sensor as a fluorescence detection tool. High coupling efficiency for fluorescence and low coupling efficiency for excitation light are key problems that need to be overcome to achieve a practical contact fluorescence imaging system. Here, we present an array of light pipe absorption filters directly connected with microchambers. The manufacturing processes are also described. Our structure makes it possible to achieve a high sensitivity that is comparable to that achieved by employing the digital ELISA with a fluorescence microscope and provides a miniaturized digital ELISA system.