A thumb-sized microscope catches images deep within active animals' brains

Researchers have succeeded in shrinking two-photon microscopy into a device that can be put on the heads of mice without inhibiting behavior.

A toy became a tool

Weijian Zong felt invincible for a moment as he peered at a glittering grid of green and blue brain cells under the microscope he had developed. "I believed that if we could get [this microscope] to function, we could accomplish anything," he says. Last March, an impromptu laboratory meeting and celebration were planned for. Zong, an optical engineer at the Kavli Institute for Systems Neuroscience in Trondheim, Norway, dressed up before presenting his recent findings to his colleagues. It was the moment his "toy became a tool," he claims.

In this case, the toy is a thumb-sized two-photon fluorescence microscope. It can illuminate and record living tissue at depths that conventional fluorescence microscopes cannot. The Mini2P, which weighs only 2.4 grams, can be attached to the head of a mouse and measure the activity of hundreds, if not thousands, of neurons while the animal runs, climbs, and leaps from a platform. Zong and his colleagues put the device through its paces in the mouse brain's vision, memory, and navigation centers, probing cells as deep as half a millimetre.

State of the Art

The instrument produces much sharper images and can capture similar numbers of cells, if not more, than head-mounted, one-photon miniscopes, which are the current state-of-the-art for in vivo imaging in freely moving animals, thanks to a custom-made lens that can follow the same cells continuously for up to one hour, or multiple times over weeks. The Mini2P offers "almost as good" resolution as a massive bench-top two-photon system, according to Fritjof Helmchen, a physicist-turned-neuroscientist at the University of Zurich in Switzerland. It's also open source, with parts lists and instructional videos on GitHub. In December, each of the 16 researchers will pay roughly €5,500 (US$5,370); The Kavli Institute in Trondheim sponsored a three-day workshop for participants to create their own two-photon miniscopes.

This opens the door to a more difficult scientific inquiry 

The Mini2P, for which Zong received this year's Tycho Jaeger Prize from the Physical Society of Norway and the Irma Salo Jger and Tycho Jgers Foundation, "opens the door to lines of scientific inquiry that were difficult, if not impossible, to initiate," according to Denise Cai, a neuroscientist at Mount Sinai's Icahn School of Medicine. And it's a development that's been in the works for years.

Fluorescence microscopy works on a simple principle: as molecules absorb energy, they get electronically excited, and when they relax, they emit light. Most microscopes are built in such a way that a single photon of light energy is enough to cause this reaction. However, in thick tissues, light is absorbed and scattered as it passes through the cellular layers. Two-photon microscopes avoid this difficulty by employing multiple, longer-wavelength photons that can penetrate deeper into tissue (two photons are required because a single longer-wavelength photon lacks the energy to activate the molecule).

Two-photon systems, on the other hand, are large and require specialized light sources and lenses. For more than two decades, researchers have sought to develop a technology that is light and compact enough to be used in freely behaving animals.

Helmchen was a forerunner. Helmchen and his colleagues developed the first portable two-photon microscope while working as a postdoctoral researcher at Bell Labs Innovations, a research and development organization in Murray Hill, New Jersey.

They published their proof-of-principle technology in 2001: an ultrafast pulsed laser coupled by a 2-meter flexible cable to a 25-gram microscope that could be put on the skull of a rat2.

The design was the first to show that a portable two-photon miniscope could record calcium signals (a visual indicator of brain activity) from individual neurons' branched projections, known as dendrites — but only in anaesthetized, head-restrained rats.

The procedure was also quite time-consuming. The researchers had to manually inject calcium-sensitive dyes into cells one at a time, then wait for the cell to light up before mounting the microscope onto the rat's head and locating the cell before attempting to capture a video. Helmchen says the scientists only imaged seven neurons over several months, recording a single cell in each trial.

A head-mounted two-photon microscope capable of imaging calcium signals in freely moving animals would require another eight years. In 2009, German researchers developed a 5.5-g portable system capable of tracking up to 20 neurons at once. They photographed neurons in rats' visual cortices that were loaded with calcium markers while the animals ran over a semicircular track3. However, Helmchen claims that the idea did not acquire traction due to the system's complexity.

Success with one photon

Zong was a second-year engineering undergraduate at Peking University in Beijing at the time. But what he truly desired — his "ultimate dream," he claims — was to "understand nature." He started his doctoral studies with biomedical engineer Heping Cheng in 2012. Cheng's lab at Peking University creates fluorescence microscopy techniques for biological study.

Single-photon miniscopes were becoming popular at the time. These gadgets, which are lightweight and sturdy enough for highly active mice, can photograph hundreds of cells at once, allowing researchers to decode entire brain circuits rather than just a few cells. They can also detect GCaMP6, an ultra-sensitive calcium sensor created following the release of the first two-photon prototypes. Single-photon miniscopes have "proven incredibly successful" in tracking behaviors such as spatial memory, song vocalization, and sleep, according to Helmchen.

According to chief commercial officer Martin Verhoef, Inscopix, a biotechnology business based in Mountain View, California, has sold over 1,500 one-photon miniscopes to more than 650 labs, with "far over 220 publications" citing work using the technology since 2011. The devices range in price from $50,000 to $150,000, depending on the setup.

A single-photon miniscope from the University of California, Los Angeles (UCLA) is an open-source alternative that comes with documentation, software, and assembly lessons on GitHub. If the pieces are purchased in bulk, the cost is roughly $500, or $1,200 as a do-it-yourself kit. UCLA miniscopes are available for around $2,000 through companies like LabMaker in Berlin and the Open Ephys Production Site in Lisbon.

According to neurologist Peyman Golshani, whose lab at UCLA helped build the UCLA miniscope (now in its fourth generation), some 500 labs around the world have utilized it since it was first built and shared around a decade ago. It has been used by researchers to study neurons that encode memories over time, for example.

Despite their capabilities, one-photon miniscopes can often only picture a few hundred micrometres deep, and the resulting fluorescence is out of focus and can blur images. That's not usually a problem in brain locations like the hippocampus, where only tiny subsets of cells fire, making the cells sparse enough to recognize in foggy images, says Edvard Moser, co-director of the Kavli Institute for Systems Neuroscience with May-Britt Moser.

However, the resolution causes an issue in the Moser lab. Grid cells are specialized neurons that hold information about location, distance, and direction. The Mosers shared the 2014 Nobel Prize in Physiology or Medicine for discovering these cells. According to May-Britt Moser, single-photon microscopy is "insufficient" for imaging grid cells. "You must have a two-photon resolution." The method necessitates the use of lasers that generate ultrafast (on the order of one millionth of a millisecond), high-power light pulses and can cost up to $200,000. "That price tag is prohibitively expensive for many research organizations," Cai says. Because two-photon systems are substantially more sophisticated than one-photon setups, creating and operating the Mini2P takes significant technical expertise.

 

However, development of the Mini2P continues in Trondheim. "We are just getting started," Zong explains after three iterations. "When a technology is publicized, it is already outdated."