Yasumi Ohta

Novel implantable imaging system for enabling simultaneous multiplanar and multipoint analysis for fluorescence potentiometry in the visual cortex

Techniques for fast, noninvasive measurement of neuronal excitability within a broad area will be of major importance for analyzing and understanding neuronal networks and animal behavior in neuroscience field. In this research, a novel implantable imaging system for fluorescence potentiometry was developed using a complementary metal-oxide semiconductor (CMOS) technology, and its application to the analysis of cultured brain slices and the brain of a living mouse is described. A CMOS image sensor, small enough to be implanted into the brain, with light-emitting diodes and an absorbing filter was developed to enable real-time fluorescence imaging. The sensor, in conjunction with a voltage-sensitive dye, was certainly able to visualize the potential statuses of neurons and obtain physiological responses in both right and left visual cortex simultaneously by using multiple sensors for the first time. This accomplished multiplanar and multipoint measurement provides multidimensional information from different aspects. The light microsensors do not disturb the animal behavior. This implies that the imaging system can combine functional fluorescence imaging in the brain with behavioral experiments in a freely moving animal.

CMOS image sensor-based implantable glucose sensor using glucose-responsive fluorescent hydrogel.

A CMOS image sensor-based implantable glucose sensor based on an optical-sensing scheme is proposed and experimentally verified. A glucose-responsive fluorescent hydrogel is used as the mediator in the measurement scheme. The wired implantable glucose sensor was realized by integrating a CMOS image sensor, hydrogel, UV light emitting diodes, and an optical filter on a flexible polyimide substrate. Feasibility of the glucose sensor was verified by both in vitro and in vivo experiments.

Functional brain fluorescence plurimetry in rat by implantable concatenated CMOS imaging system

Measurement of brain activity in multiple areas simultaneously by minimally invasive methods contributes to the study of neuroscience and development of brain machine interfaces. However, this requires compact wearable instruments that do not inhibit natural movements. Application of optical potentiometry with voltage-sensitive fluorescent dye using an implantable image sensor is also useful. However, the increasing number of leads required for the multiple wired sensors to measure larger domains inhibits natural behavior. For imaging broad areas by numerous sensors without excessive wiring, a web-like sensor that can wrap the brain was developed. Kaleidoscopic potentiometry is possible using the imaging system with concatenated sensors by changing the alignment of the sensors. This paper describes organization of the system, evaluation of the system by a fluorescence imaging, and finally, functional brain fluorescence plurimetry by the sensor. The recorded data in rat somatosensory cortex using the developed multiple-area imaging system compared well with electrophysiology results.

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.

Implantable CMOS imaging device with absorption filters for green fluorescence imaging

Green fluorescent materials such as Green Fluorescence Protein (GFP) and fluorescein are often used for observing neural activities. Thus, it is important to observe the fluorescence in a freely moving state in order to understand neural activities corresponding to behaviors. In this work, we developed an implantable CMOS imaging device for in-vivo green fluorescence imaging with efficient excitation light rejection using a combination of absorption filters. An interference filter is usually used for a fluorescence microscope in order to achieve high fluorescence imaging sensitivity. However, in the case of the implantable device, interference filters are not suitable because their transmission spectra depend on incident angle. To solve this problem we used two kinds of absorption filters that do not have angle dependence. An absorption filter consisting of yellow dye (VARYFAST YELLOW 3150) was coated on the pixel array of an image sensor. The rejection ratio of ideal excitation light (490 nm) against green fluorescence (510 nm) was 99.66%. However, the blue LED as an excitation light source has a broad emission spectrum and its intensity at 510 nm is 2.2 x 10-2 times the emission peak intensity. By coating LEDs with the emission absorption filters, the intensity of the unwanted component of the excitation light was reduced to 1.4 x 10-4. Using the combination of absorption filters, we achieved excitation light transmittance of 10-5 onto the image sensor. It is expected that high-sensitivity green fluorescence imaging of neural activities in a freely moving mouse will be possible by using this technology.

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.

Implantable self-reset CMOS image sensor and its application to hemodynamic response detection in living mouse brain

A self-reset pixel of 15 × 15 µm2 with high signal-to-noise ratio (effective peak SNR 64 dB) for an implantable image sensor has been developed for intrinsic signal detection arising from hemodynamic responses in a living mouse brain. For detecting local conversion between oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) in brain tissues, an implantable imaging device was fabricated with our newly designed self-reset image sensor and orange light-emitting diodes (LEDs; λ = 605 nm). We demonstrated imaging of hemodynamic responses in the sensory cortical area accompanied by forelimb stimulation of a living mouse. The implantable imaging device for intrinsic signal detection is expected to be a powerful tool to measure brain activities in living animals used in behavioral analysis.

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.

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.