The 83rd JSAP Autumn Meeting 2022 Highlighted Presentations
October 24, 2022

Head-mounted CMOS imaging device for blood flow and fluorescent measurement

Contributing to the development of brain science anddrug discovery by developing an ultra-compact CMOS
imaging device that can simultaneously measure
cerebral blood flow and neural activity
Nara Advanced Institute of Science and Technology (1), Kyushu University (2)Makoto Haruta (1), Mark Christian Guinto (1), Yasumi Ota (1), Asumiko Kawahara (1), Kosei Takehara (1), Yoyo Tashiro (1),(2),Kiyotaka Sasakawa (1), Jun Ota (1)summary• By placing a 0.5-g ultra-compact imaging device on the surface of the mouse brain,
researchers succeeded in measuring blood flow and neuronal activity almost
simultaneously under free-running conditions without damaging the brain, which had
been a difficult feat to achieve.
• Using a device that can observe the brain surface while alternately illuminating green andblue LED light sources, they measured changes in the amount of hemoglobin in theblood, including changes in the calcium fluorescence (*1) content of mice geneticallyengineered to express GCaMP.
• The simultaneous measurement of blood flow and neural activity will be beneficial in theresearch on the pathogenic mechanism of diseases such as cerebral infarction andmigraine, including brain functions via behavior, leading to the development of advanced
technologies such as drug discovery and brain-machine interfaces.
Makito Haruta et al. developed an ultra-compact CMOS imaging device at the Nara Institute of Science andTechnology (NAIST), to observe detailed brain activity in freely behaving mice. This technology is a lens-lessimaging method that enables the simultaneous measurement of blood flow and fluorescence, to elucidate
the relationship between blood flow and neural activity in the brain. By utilizing this device, it is now possible
to study mouse and brain activities from an unprecedented angle. This device is expected to be used toelucidate the mechanisms of diseases related to cerebral blood flow and neural activity, such as cerebral
infarction and migraine, for drug discovery and to elucidate brain functions via the detailed measurement ofbrain activity under free-running conditions.

detail

Novel device for measuring the active brain in real timeWhen the brain is active, the blood flow in the active region increases. Therefore, cerebral
blood flow is an important indicator of brain activity and condition. Blood flow is also
closely related to brain pathology. When blood flow is reduced owing to the narrowing ofblood vessels, brain function is impaired. Several diseases are related to cerebral bloodflow, such as cerebral infarction and migraine headaches.
PET and fMRI are conventionally adopted to measure brain activity in humans. For studies
with animals, in addition to methods based on the same principles as those used forhumans, there are also methods that adopt fluorescence microscopy; nevertheless, it is
crucial to perform measurements under anesthesia or with the head immobilized bytraining or other means. Regardless, the animal must be immobilized under anesthesia ortraining, and the animal's head must be immobilized. Under such conditions, theexperimental animal is subjected to unnecessary stress, and the brain activity to beobserved cannot be considered pure.
To address this problem, the research group developed an ultra-compact imaging device
that combines a CMOS image sensor and LED, and by implanting it in the head of amouse, established a technology that enables the long-term observation of brain activity
without placing a burden on the mouse.
Specifically, using green light, which is strongly absorbed by hemoglobin (*2), as a lightsource, blood vessels on the surface of the brain can be observed with a CMOS image
sensor. In addition to enabling the clear observation of minute blood vessels, thistechnology also enables the measurement of blood flow and blood flow velocity.
A blue LED adopted as a fluorescence excitation light source and a light absorption filter(long-pass filter) on the image sensor were utilized to detect changes in the fluorescenceintensity of GCaMP, which is a fluorescent calcium probe used to observe neural activity.
Elements other than the blood flow can be observed simultaneously by alternately turningthese two light sources. At (the Japan Society of Applied Physics Autumn Meeting), Dr.
Haruta presented the results of a study on the relationship between blood flow and direct
neuronal activity together with calcium fluorescence imaging in mice geneticallyengineered to express GCaMP.
According to Haruta, "this device is equipped with LEDs for blood flow observation andfluorescence excitation. The device is equipped with LEDs for blood-flow observation andfluorescence excitation. Because blood flow measures endogenous signals, it can bemeasured without staining. In addition, we attempt to simultaneously measure blood flowand neural activity by combining the measurement with genetically engineered mice withfluorescent probes, which has already been established.”

The range of applications can be expanded by altering the combination of light mounted
on the device and the genes expressed in the mice. For example, it is possible to combine
optogenetics, which induces or inhibits neural activity via optical stimulation, with theobservation of blood flow or measurement of neural activity.

Simultaneous Measurement of Blood Flow and Nerve Activity Opens the Possibilityof Drug Discovery
This device, which is based on the fusion of different fields of neuroscience andsemiconductor engineering, adopts the semiconductor packaging technology, biomedical
implantation technology used in the biomedical field, and fluorescence microscopy andfluorescence imaging technology, using genetically engineered mice that have beencultivated in the neuroscience field. By altering the size of the device, it is possible toadjust the area of the brain that can be measured, and observe the interaction of multiple
areas.
According to Haruta, "our greatest advantage is that we can design the image sensor itself
and fabricate a device that fits the research."
Recently, significant progress has been made in brain research owing to technologicaldevelopments. Haruta et al. 's novel imaging device is a groundbreaking technology thatcould inspire brain scientists to experiment, connect, and advance several previously
unproven findings.
The results of this study will be utilized in the development of novel therapies and drug
discovery. It can also be adopted to examine the pharmacokinetics and effects of drugs
during development. In particular, the ability to measure blood flow velocity can provide
valuable data for the development of more effective drugs.
According to Haruta, "this technology will allow us to get closer to the mechanisms of
diseases involving cerebral blood flow and neurological activity. We are currently
conducting joint research on cerebral infarction and migraines. In addition, because wecan comprehensively observe brain activity in mice under free-running conditions, webelieve that studying the relationship between behavior, blood flow, and neuronal activity
could benefit technologies such as brain-machine interfaces.
annotations*!Calcium fluorescence imaging of mice expressing GCaMP
GCAMP binds to calcium and emits fluorescence. When neurons are active, calcium flows into the cells. Therefore, byexpressing GCAMP in mouse neurons via genetic recombination, it is possible to directly observe neuronal activity bymeasuring the fluorescence emitted by GCaMP.
*2 Absorption spectrum of hemoglobin
Red blood cells are associated with hemoglobin. Because hemoglobin absorbs light of a specific wavelength, the amountof hemoglobin can be determined, and blood flow can be estimated by shining light on it and measuring the amount oflight absorbed by the hemoglobin.

Figures

Conventional imaging systems that utilize lenses require the mouse to be immobilized; however, this technology canmeasure brain activity in free-moving mice (top left). The device was fixed on the dura mater without skull removal,
thereby allowing imaging without damaging the brain (upper right panel). Even minute blood vessels can becomprehensively observed (bottom right panel).