Masahiro Nunoshita

Real time in vivo imaging and measurement of serine protease activity in the mouse hippocampus using a dedicated complementary metal-oxide semiconductor imaging device.

The aim of the present study is to demonstrate the application of complementary metal-oxide semiconductor (CMOS) imaging technology for studying the mouse brain. By using a dedicated CMOS image sensor, we have successfully imaged and measured brain serine protease activity in vivo, in real-time, and for an extended period of time. We have developed a biofluorescence imaging device by packaging the CMOS image sensor which enabled on-chip imaging configuration. In this configuration, no optics are required whereby an excitation filter is applied onto the sensor to replace the filter cube block found in conventional fluorescence microscopes. The fully packaged device measures 350 microm thick x 2.7 mm wide, consists of an array of 176 x 144 pixels, and is small enough for measurement inside a single hemisphere of the mouse brain, while still providing sufficient imaging resolution. In the experiment, intraperitoneally injected kainic acid induced upregulation of serine protease activity in the brain. These events were captured in real time by imaging and measuring the fluorescence from a fluorogenic substrate that detected this activity. The entire device, which weighs less than 1% of the body weight of the mouse, holds promise for studying freely moving animals.

A complementary metal-oxide-semiconductor image sensor for on-chip in vitro and in vivo imaging of the mouse hippocampus

This paper describes the development of a complementary metal–oxide–semiconductor (CMOS) image sensor for in vitro and in vivo imaging of the hippocampus. The 176×144 pixel array image sensor is designed based on a modified three-transistor active pixel sensor circuit. Flexibility in readout for real-time imaging and wide dynamic range measurement is implemented using analog and digital output. A minimum light intensity detection level of 50 nW/cm2 has been measured using the image sensor. A novel packaging method is developed to enable both in vitro and in vivo imaging. In this method, a color filter is applied onto the image sensor that selectively blocks excitation light transmittance to below -44 dB. The packaged device thickness measuring 350 µm, limits tissue damage during invasive imaging. Using the device, static images of the mouse brain slice and real time imaging of the hippocampus of a mouse are successfully demonstrated for the first time.

Pulse modulation CMOS image sensor for bio-fluorescence imaging applications

For wide dynamic range, compatibility with digital circuits, and low-voltage operation, the pulse modulation technique is suitable for an implanted bioimage sensor. We demonstrate bio-fluorescence imaging of the hippocampus in a sliced mouse brain using a pulse modulation-based image sensor. The sensor architecture and system configuration are discussed. In addition, we develop an imaging device for implantation into a mouse brain in order to measure the neural activity in the hippocampus. The device is composed of a pulse modulation image sensor with 128/spl times/128 pixels and a fiber illuminator on a polyimide substrate.

Development of a Fully Integrated Complementary Metal–Oxide–Semiconductor Image Sensor-Based Device for Real-Time In vivo Fluorescence Imaging inside the Mouse Hippocampus

In our previous work, we demonstrated the potential of a complementary metal–oxide–semiconductor (CMOS) imaging device for use in imaging of the mouse brain. We showed that the device is capable of detecting fluorescence signal inside the mouse brain and successfully imaged real-time protease activity inside the hippocampus. In this work, we have improved the imaging device by integrating an excitation light source in the form of an ultraviolet light-emitting diode chip and an injection needle onto the sensor chip. This results in a compact single device imaging system for minimal invasive imaging inside the mouse brain. Also experimental repeatability is improved which enabled us to successful perform calibration of fluorophore concentration using the device. Fluorescence imaging experiments inside the brain phantom as well as in the mouse brain show that the device is capable of real time fluorescence detection. Using the device, we found that diffusion rate of chemical injected into the brain is smaller than 10 pmol/min. This work is expected to lead to the successful use of a CMOS imaging device for the study of the functions of the brain.

Wide dynamic range pulse modulation image sensor for on-chip bioimaging applications

Image sensors with a pulse modulation measurement scheme are fabricated for bioimaging and biosensing applications. We designed pulse modulation photosensors and a 64/spl times/64-pixel image sensor. We demonstrated the feasibility of the pulse modulation measurement scheme for biosensing applications. We obtained a dynamic range of 120 dB and minimum sensing intensity level of 2 nW/cm/sup 2/. We also confirmed that 0.2% of intensity change is detectable at the minimum intensity region. An in-vitro, on-chip imaging of a mouse hippocampus was successfully demonstrated.

Development of a CMOS Imaging Device for Functional Imaging Inside the Mouse Brain

We propose the use of a CMOS image sensor for in vivo functional imaging of the mouse brain. A dedicated image sensor with 176 times 144 pixels array is designed, fabricated, and packaged to enable on-chip fluorescence imaging. Using this configuration, we have successfully performed imaging inside the intact mouse brain. Furthermore, we have demonstrated functional imaging inside the mouse brain. This is done in conjunction with a fluorogenic substrate, which detects the presence of serine protease inside the brain. By imaging the fluorescence signal upon injection of a stimulant, we have successfully measured the brain protease activity and accurately determined its reaction onset, which is close to 1 hr 28 min after injection of the stimulant. This method represents a new approach for neural imaging using an inexpensive CMOS imaging device

Pulse modulation image sensors for on-chip bioimaging and biosensing applications

Image sensors with pulse modulation measurement scheme are fabricated for bioimaging and biosensing ap-plications. We designed pulse modulation photosensors, a 64×64-pixels image sensor for in vitro bioimaging, and a 176×144-pixels (QCIF) image sensor for in vivo bioimaging. We demonstrated the feasibility of the pulse modulation measurement scheme for biosensing applications. We obtained a dynamic range of 120dB and minimum sensing intensity level of 2nW/cm2. We also confirmed that 0.2% of intensity change is detectable at the minimum intensity region. An in vitro, on-chip imaging of a mouse hippocampus was successfully demonstrated. A sensor module for in vivo imaging is also developed.