Yoshinori Sunaga

An implantable CMOS device for blood-flow imaging during experiments on freely moving rats

An observation technique for animal brain activity under freely moving conditions is important to understand brain functions because brain activity under an anesthetized condition is different from that under a nonanesthetized condition. We have developed an ultrasmall CMOS imaging device for brain activity observation under freely moving conditions. This device is composed of a CMOS image sensor chip and nine LEDs for illumination. It weighs only 0.02 g and its small size enables experiments to be performed without restricting animal movement. This feature is advantageous for brain imaging, particularly in freely moving situations. In this study, we have demonstrated blood-flow imaging using the device for the stable observation of brain activity over a long period. The blood flow can be observed without staining the brain during optical imaging. We have successfully estimated the blood-flow velocity under freely moving conditions.

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 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.

Intrinsic signal imaging of brain function using a small implantable CMOS imaging device

A brain functional imaging technique over a long period is important to understand brain functions related to animal behavior. We have developed a small implantable CMOS imaging device for measuring brain activity in freely moving animals. This device is composed of a CMOS image sensor chip and LEDs for illumination. In this study, we demonstrated intrinsic signal imaging of blood flow using the device with a green LED light source at a peak wavelength of 535 nm, which corresponds to one of the absorption spectral peaks of blood cells. Brain activity increases regional blood flow. The device light weight of about 0.02 g makes it possible to stably measure brain activity through blood flow over a long period. The device has successfully measured the intrinsic signal related to sensory stimulation on the primary somatosensory cortex.

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.

Needle type CMOS imaging device for fluorescence imaging of deep brain activities with low invasiveness

We propose a thin type CMOS image sensor for measuring neural activities. The sensor is designed very thin shape for low invasiveness to a mouse brain. We demonstrate fluorescence imaging with the sensor.

Demonstration of implantable CMOS image sensors for functional brain imaging

We have developed implantable complementary -metal-oxide-semiconductor (CMOS) imaging devices for brain functional imaging (Fig.1) [1]. With the device, it is possible to perform fluorescence or absorption imaging and observe various neural activities of the mouse brain in a freely moving state [2, 3]. The device comprises a CMOS image sensor, and LEDs as excitation or illumination light sources. The pixel array is coated with absorption filter for excitation light rejection in the case of fluorescence imaging. It is expected that brain imaging in a freely moving mouse will be possible to elucidate relations between neural activities and behaviors.

An implantable image sensor with self-reset function for brain imaging

We designed and fabricated an implantable image sensor high peak signal-to-noise ratio (SNR). In bio-imaging, it is often necessary to observe small changes of fluorescence, absorption, scattering. The peak SNR of a image sensor is limited by the shot-noise, which is proportional to the square root of incident light intensity. In this study, we avoid pixel saturation by self-reset function integrated in a pixel and increase the peak SNR. The pixel size is 15μm × 15μm and the peak SNR is over 60 dB We also demonstrated blood flow imaging of a rat brain surface by using the sensor.