Various techniques for image color reproducibility under low-light conditions have been proposed, such as high sensitivity, a combination of visible (VIS) and infrared (IR) light, and coloring monochromatic images. However, when the illuminance falls below a certain level, color images cannot be obtained without prior color information. Previously, the exclusive use of IR without VIS illumination was proposed to achieve a pseudocolor image (basic IR color). It improved visibility compared with conventional monochrome images. However, there are cases, depending on the objects, when basic IR light cannot reproduce the correct color. An image processing method for enhancing color reproducibility is proposed, particularly for objects that are not suitable for the basic IR color (enhanced IR color). Moreover, we developed an algorithm to combine the advantages of both VIS and IR colors by utilizing signals of six wavelengths: three wavelengths each of VIS and IR. The proposed method includes an automatic transition between the optimal combination of VIS and IR colors when the illumination level changes, thus providing images with superior color reproducibility under various illumination levels compared with basic IR color.
Significance: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings.
Aim: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions.
Approach: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λcenter: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions.
Results: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed.
Conclusions: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.
Significance: Gene expression analysis is an important fundamental area of biomedical research. However, live gene expression imaging has proven challenging due to constraints in conventional optical devices and fluorescent reporters.
Aim: Our aim is to develop smaller, more cost-effective, and versatile imaging capabilities compared with conventional devices. Bioluminescence reporter-based gene expression analysis was targeted due to its advantages over fluorescence-based imaging.
Approach: We created a small compact imaging system using micro-CMOS image sensors (μCIS). The μCIS model had an improved pixel design and a patterned absorption filter array to detect the low light intensity of bioluminescence.
Results: The device demonstrated lower dark current, lower temporal noise, and higher sensitivity compared with previous designs. The filter array enabled us to subtract dark current drift and attain a clearer light signal. These improvements allowed us to measure bioluminescence reporter-based gene expression in living mammalian cells.
Conclusion: Using our μCIS system for bioluminescence imaging in the future, the device can be implanted in vivo for simultaneous gene expression imaging, behavioral analysis, and optogenetic modulation.
An implantable image sensor device is extremely small and lightweight owing to the use of a specially designed complementary metal oxide semiconductor (CMOS) image sensor. It is intended to be applied in in-vivo brain function imaging under free behavior of an observation target such as a mouse. Because of its low invasiveness, it is suitable for the simultaneous imaging of the deep brain along with several other regions of the brain, and for long-term observations. In order to realize low invasiveness, it is required to perform contact imaging without using a lens. This gives rise to a different set of challenges from that faced by microscope systems that use a general lens. In this study, we developed a light source suited for the in-vivo implantation of a fluorescence imaging system to facilitate green fluorescence observation. A thinned InGaN-based blue LED was used as an excitation light source. Normally, an LED has an emission bandwidth that is too broad for fluorescence imaging. In the proposed device, in addition to the interference filter, absorption layers are added in order to remove the unwanted light emission component by using a different strategy. The interference filter used was obtained by transferring a long-pass filter formed on a quartz glass filter image onto a fiber optic plate (FOP) with a low numerical aperture (NA) by the laser lift-off (LLO) method. The interference filter showed sufficiently high performance as an excitation filter even after it was transferred to the light source. The absorption layers composed of a low-NA FOP and a blue absorption layer are used to remove unwanted components. Light rays with a high incident angle, leak through the interference filter when the absorption layers are not present. On the other hand, the unwanted components in the emission bandwidth were successfully reduced in the proposed structure.
The emission filter plays a key role in resolving a modest-quality image of the lens-free fluorescent imager. The complementary structure of an interference filter and absorption filters exhibits a high-rejection ratio, corresponding to the lens-based fluorescence device. However, existing fabrication methods are facing challenges to reach a reasonable filter thickness for low invasiveness. It is difficult to deposit interference filters on the polymer-based absorption filter and CMOS die directly. Conversely, the interference is fragile and easy to crack so that transferring from its substrate to the image sensor is a fatiguing task. Here we report composite filter fabrication using laser lift-off (LLO) and silicon plasma etching. The LLO utilized high energy laser to separate the interference filter from the glass substrate, whereas the plasma etching tailored SiF6 gas to completely annihilate silicon-substrate whereby the filter was deposited beforehand. As a result, a narrow-size filter is successfully fabricated by LLO, yet a crack issue for a larger sensor size remains unsolved. On the other hand, the plasma etching produced large-size and spotless filters with relatively high reproducibility. Additionally, this method offers multiple device fabrication in a single process, which, we expect, could intensify largescale lens-free fluorescent imager applications in the future.
Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and
viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small
volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent
microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom
image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470
nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber
array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with
antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with
the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the
glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image
processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads
with collimated illumination are observed. By varying the incident angle and image composition, microbeads were
successfully imaged.
The structures in advanced complementary metal-oxide-semiconductor (CMOS) integrated circuit technology are in the range of deep-submicron. It allows designing and integrating nano-photonic structures for the visible to near infrared region on a chip. In this work, we designed and fabricated an image sensor with on-pixel metal wire grid polarizers by using a 65-nm standard CMOS technology. It is known that the extinction ratio of a metal wire grid polarizer is increased with decrease in the grid pitch. With the metal wire layers of the 65-nm technology, the grid pitch sufficiently smaller than the wavelengths of visible light can be realized. The extinction ratio of approximately 20 dB has been successfully achieved at a wavelength of 750 nm. In the CMOS technologies, it is usual to include multiple metal layers. This feature is also useful to increase the extinction ratio of polarizers. We designed dual layer polarizers. Each layer partially reflects incident light. Thus, the layers form a cavity and its transmission spectrum depends on the layer position. The extinction ratio of 19.2 dB at 780 nm was achieved with the grid pitch greater than the single layer polarizer. The high extinction ratio is obtained only red to near infrared region because the fine metal layers of deepsubmicron standard CMOS process is usually composed of Cu. Thus, it should be applied for measurement or observation where wide spectrum is not required such as optical rotation measurement of optically active materials or electro-optic imaging of RF/THz wave.
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.
The oscillation wavelength of a Nd3+-doped tellurite glass microsphere laser is controlled using a λΑ/4-shifted grating fabricated on the surface of the microsphere. The lasing wavelength is in agreement with the high transmission wavelength of the grating estimated from the grating period and the effective index of the whispering gallery mode.
The authors have successfully demonstrated continuous-wave oscillation in a Nd-doped tellurite glass microsphere laser at 1.06 μm for the first time. Microspheres with diameters of 50 to a few hundred micrometres are fabricated by melting using an electric heater. Emission spectra reveal that the devices exhibit resonances corresponding to whispering gallery modes. Lasing threshold of pump power was about 5 mW.
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