Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue

Abstract

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.

Keywords: (110.2970) Image detection systems; (170.2655) Functional monitoring and imaging; (280.1415) Biological sensing and sensors.

Fig. 1

Schematics of epi-illumination and imaging in brain tissues using a light-emitting diode (LED) and a semiconductor imaging device.

Fig. 2

Schematics and photographs of the implantable semiconductor imaging device. (a) Schematics of the implantable semiconductor imaging device. The device is constructed with a thin-film absorption filter, an imaging sensor chip, and a flexible printed circuit (FPC). (b) Photograph of the implantable semiconductor imaging device. The semiconductor image sensor chip is immobilized onto the FPC, which is connected to the small relay board. (c) Fabrication process of the implantable semiconductor imaging device.

Fig. 3

Schematics and photographs of fabricated CMOS image-sensor chip. (a) The CMOS imaging-sensor chip has a pixel array and four bonding pads. The imaging sensor chip is 450-μm wide, 1500-μm long, and 150-μm thick. (b) Block diagram of the CMOS image-sensor chip. The chip has three inputs (V_DD, GND, and CLK) and one output (Out). Schematics of output circuit was reported in detail elsewhere [44]. (c) Sensitivity of the CMOS image-sensor chip.

Fig. 4

Performance of thin-film absorption filters and the semiconductor imaging device integrated with the thin-film absorption filter. (a) Transmittance T at 470 nm as a function of film thickness t (μm) of the absorption filter. The film thickness was controlled by the numbers of spin-coated layers. Horizontal error bars show the standard deviations of the thickness. (b) The transmittance spectra of the absorption filters of thickness 0.8 μm and 3.4 μm. (c) Obtained fluorescent image using the image sensor integrated with the thin-film absorption filter. Fluorescent microspheres (ϕ = 15 μm) were placed directly onto the image sensor and illuminated by excitation light using a blue LED. Representative fluorescent intensity profile along x-pixels was shown at the upper-right corner. The experimental data (dots) were fitted by a Gaussian function (line).

Fig. 5

Numerical analysis of the intensity distribution of excitation light in brain tissues. (a) Cross-sectional image of excitation light intensity in brain tissues. Contours represent the surfaces where the light intensity drops to 10%, 1%, and 0.1%. (b) Normalized excitation light intensity on the surface of the image sensor at various positions (L = 0, 1, and 2 mm). (c) Experimentally measured light intensity profiles (dots) and computationally calculated light intensity profiles (lines) at various positions (L = 0, 1, and 2 mm) as a function of y-pixels.

Fig. 6

Fluorescence imaging in deep brain tissues using the implantable semiconductor device. (a) Photographs of the exposed brain surface of a mouse and illuminated brain surface using an LED. (b) Obtained images of fluorescent microspheres (ϕ = 15 μm) in deep brain tissue (1–2 mm in depth) by implanted image sensors at various positions (L = 0, 1, and 2 mm). Saturation of fluorescent signal in the top sensor area (y-pixel > 300 μm) was observed with the image sensor at L = 0 mm. Elliptical shadowing in the images was considered to be an artifact caused by a silicone rubber sheet used to attach microspheres on the device. (c) The ratio of the fluorescent intensity of microspheres in the top sensor area (y-pixel > 300 μm) and bottom sensor area (y-pixel < –300 μm). The intensity ratio of the image sensor at L = 2 mm was at the same level as that of a control image obtained under uniform illumination in vitro. Error bars show the standard deviations.

Fig. 7

Fluorescence imaging of fluorescent substances embedded in brain tissues obtained using the implantable semiconductor device. (a) Obtained images of fluorescent microspheres (ϕ = 15 μm) embedded in a 50-μm-thick brain tissue slice. (b) Representative fluorescent intensity profile along x-pixels of the microsphere embedded in brain tissue slice. The experimental data (dots) were fitted by a Gaussian function (line). (c) Fluorescence signal change of blood vessels embedded in the brain tissue of a living mouse that was labeled with quantum dots.