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A Mini Microscope Implanted in Brain of Freely Moving, Unanesthetized Mice Permits Weeks of Imaging

Article In Brief

A research team was able to observe the progression of a brain tumor and its microvasculature in mice, imaging continuously over 16 days, thanks to the advance of a miniature microscope capable of in vivo brain imaging in freely moving or awake mice. The device has the potential to improve real-time image of pathology related tto stroke, tumors, epileptic seizures, and other brain disorders.

A microscope as big as a fingertip, weighing no more than a penny, has been developed by a team at Johns Hopkins and implanted over the brain of unanesthetized, freely moving mice to permit weeks of functional imaging.

The device, described in a paper January 9 in Nature Communications, offers investigators a new window—literally—into how the brain and its vasculature respond to stroke, tumors, epileptic seizures, and other brain disorders.

Among its unique capabilities is the combination of three light channels: a fluorescence channel for imaging neural activity, an intrinsic optical channel for imaging hemoglobin absorption, and laser speckle contrast for imaging perfusion. Previous head-mounted devices have incorporated only a single optical modality.

While offering a wide field of view capable of viewing the entire cortical surface, its magnification can accurately resolve down to 0.5 μm, or 0.00002 of an inch. Because the base and sensor are produced with a 3D printer to fit each mouse's head, and the optoelectronic components are bought off-the-shelf, the device costs a fraction of currently available multi-contrast benchtop imaging systems, according to the paper.

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“My expectation is that this microscope still has to go through some cycles of improvement and demonstrate additional widespread applications before commercialization.”

—DR. NITISH V. THAKOR

“We believe in the near future this microscope's affordability and flexibility will play a major role in making it a ubiquitous laboratory tool,” the authors of the paper wrote.

Two physicians familiar with the paper agreed that the device represents an important advance, calling it “extraordinary” and “remarkable,” while two other scientists who conduct similar research were more restrained in their characterization. But all agreed that the ability to longitudinally image the brain of freely behaving, un-anesthetized mice can shed light on many unanswered questions in neurology.

“Many neurology researchers rely on techniques developed decades ago—looking at ex-vivo tissues using histology and immunohistochemistry,” said Elizabeth M.C. Hillman, PhD, professor of biomedical engineering and radiology at Columbia University's Mortimor B. Zuckerman Mind Brain Behavior Institute. “There are many new tools for interrogating the living brain, which could offer so many important insights into brain disease and dysfunction.”

Study Details

Each customized, disposable head mount for the microscope is attached permanently over a surgically prepared cranial window. Screws within the head mount permit the microscope to be attached or detached as needed. Although the entire device actually weighs nine grams, an overhead tether reduces the amount of weight on the mouse's head to just three grams. Measures of blood corticosterone levels, as well as videos of animal behavior, showed that they gradually became habituated to the device and moved about in a natural manner.

The Hopkins team validated the microscope's performance against a standard benchtop imaging system and found it comparable. The device's 640 x 640 pixel sensor acquired images at 15 frames per second.

To determine the device's neural and hemodynamic response to auditory stimulation, the investigators performed multi-contrast imaging of the mouse auditory cortex, successfully capturing the time evolution of neural activation (via calcium fluorescence) and the accompanying hemodynamic response. As expected, the sharp rise in calcium accumulation due to neural firing was followed by increases in total hemoglobin and cerebral blood flow, and a decrease in deoxyhemoglobin.

The study authors, led by senior author Arvind P. Pathak, PhD, also demonstrated the device's synchronization with EEG during anesthesia recovery.

In the paper's most dramatic finding, the progression of a brain tumor and its microvasculature was imaged continuously over 16 days. After the mouse brain was inoculated with a tumor seed, the investigators observed the formation of a new “mother” blood vessel migrating toward it, followed by angiogenic sprouting from that parent vessel to support tumor growth.

A coauthor of the paper said it was “remarkable” that mice fitted with the microscope ignore it. “Remarkably, the rodents accept the head-mounted microscope, so long as it is light in weight,” said Nitish V. Thakor, PhD, professor of biomedical engineering and professor of neurology at Hopkins. At the low weight of the device implanted into their skull, “They seem not to care.”

For now, the microscope, designed by first author Janaka Senarathna, PhD, has been shown to work for no more than a few weeks. “We weren't pushing for months or years,” he said. “We wanted to study the evolution of tumors, strokes, epilepsy. We can also study behavior and learning in a maze. Those require up to a few weeks.”

He and three other authors of the paper have an international patent pending on the microscope. But, Dr. Thakor said, “My expectation is that this microscope still has to go through some cycles of improvement, and demonstrate additional widespread applications before commercialization.”

Expert Commentary

Dr. Hillman, who has been working in the field of optical brain imaging for some two decades, noted that the device described in Dr. Thakor's paper images only the brain's surface, not deeper structures such as the hippocampus, which are the focus of several other head-mounted microscopes that require a special lens to be physically inserted into the brain.

However, she added, “This surface imaging is advantageous if you want to study the brain's vasculature, which is predominantly on the cortical surface and is easily damaged. In my own work, we tend to have the animals head-fixed. This new device would allow us to let the animals do a wider range of behaviors in a more natural setting.”

The capacity to image an awake, freely moving animal was also welcomed by Theodore H. Schwartz, MD, FACS, the David and Ursel Barnes professor of minimally invasive neurosurgery and director of the Epilepsy Research Laboratory at Weill Cornell Medicine.

“Our lab does similar studies, but the animals are not freely moving, so this technology overcomes that obstacle,” Dr. Schwartz said. “That's really exciting. It's never been done before on a macroscopic scale. That's a big deal.”

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“Combining such high resolution imaging using multiple functional modalities is a powerful addition for non-invasive, in vivo longitudinal studies of the brain. This is an engineering tour de force with profound capabilities.”

—DR. BARRY KOSOFSKY

In epilepsy research, he said, it's impossible to keep an animal or human in an fMRI for the hours or days necessary to observe a naturally occurring seizure.

“The only way to do a study like that is to have an animal that's awake and running around, and then to record brain changes indefinitely with a variety of markers,” Dr. Schwartz said. “You'll be able to image changes that precede the seizure, and then see where the seizure starts and how it spreads. If we could show that 20 minutes before the seizure starts there is a clear, discernible alteration in certain neurons' activity or blood flow, perhaps then we could intervene.”

Barry Kosofsky, MD, PhD, the Goldsmith Foundation professor of pediatrics, neurology and neuroscience, and radiology, and director of the Horace W. Goldsmith Foundation Laboratory of Molecular and Developmental Neuroscience at Weill Cornell Medicine, called the new device “extraordinary.”

“Combining such high resolution imaging using multiple functional modalities is a powerful addition for non-invasive, in vivo longitudinal studies of the brain,” he said. “This is an engineering tour de force with profound capabilities.”

He noted, however, that the tether attached to an overhanging suspension would prevent mice from doing some of the tasks his laboratory runs, including water-based behavioral assays such as the Morris water maze and the tail-suspension test.

Figure

“Our lab does similar studies, but the animals are not freely moving, so this technology overcomes that obstacle. That's really exciting. It's never been done before on a macroscopic scale. That's a big deal.”

—DR. THEODORE H. SCHWARTZ

Timothy H. Murphy, professor of neuroscience at the University of British Columbia, agreed that the tether could be an obstacle for certain studies.

“It still looks like a huge device to me,” Dr. Murphy said. “The suspension rig is bulky. It's definitely going in the right direction, but it's an incremental advance. What we would really like to see is something that is 3 grams or less without a cable.”

Even so, Dr. Murphy emphasized, “This group at Hopkins is really pushing the boundaries. Even at its current weight, this is a cool advance that supports high-quality multi-modal imaging.”

Dr. Murphy published a paper in Nature Communications in June of 2016 describing another approach to imaging the cortex of freely moving mice in their home cage. His group trained mice to periodically stick their heads, already prepared with a cranial window, into an aperture that briefly locks them in place and permits brief imaging of the brain. The unsupervised mice initiated over 7,000 imaging sessions over the course of 90 days.

Disclosures

Drs. Thakor, Kosofsky, and Murphy had no disclosures. Dr. Schwartz disclosed he has served as a consultant to Integra.

Link Up for More Information

• Senarathna J, Yu H, Deng C, et al. A miniature multi-contrast microscope for functional imaging in freely behaving animals https://www.nature.com/articles/s41467-018-07926-z. Nat Commun 2019;10(1):99.
    • Park JH, Platisa J, Verhagen JV, et al. Head-mountable high speed camera for optical neural recording https://www.ncbi.nlm.nih.gov/pubmed/21763348. J Neurosci Methods 2011;201(2):290–295
      • Ma Y, Shaik MA, Kim SH, et al. Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches https://royalsocietypublishing.org/doi/full/10.1098/rstb.2015.0360. Philos Tran R Soc Lond B Biol Sci 2016;371(1705). pii: 20150360.
        • Murphy TH, Boyd JD, Bolaños F, et al. High-throughput automated home-cage mesoscopic functional imaging of mouse cortex https://www.nature.com/articles/ncomms11611. Nat Commun 2016;7:11611