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Sarah Webb, Ph.D.
BioTechniques, Vol. 61, No. 6, December 2016, pp. 285–291
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Microscopy continues to expand our knowledge of brain structure and function. Sarah Webb looks at some of the latest tools and techniques leading the charge.

over the last 20 years, neuroscientists have discovered that many neurons in the body spontaneously fire in groups. “We’re pretty certain that these neuronal ensembles exist in the brain, but we still don’t know what they do,” says Rafael Yuste of Columbia University. While traditional optical microscopy paired with tools such as calcium imaging can follow active neurons, what these neurons are actually doing within the brain is still a mystery.

Yuste is keen on solving this mystery. His group has focused recently on new techniques that can write neuronal activity, such as optogenetics and optochemistry, as well as complementary microscopy techniques to observe the experimental results. Recently, they used two-photon methods to write firing patterns into groups of neurons in live mice and then monitored the activity based on calcium signaling to connect those patterns with behavior. As a result, the team successfully imprinted new neuronal circuits in the brain of a mouse by simultaneously stimulating those neurons and testing whether the artificial patterns interfere with a specific behavior—licking for juice in response to a pattern of horizontal or vertical lines (1). Interestingly, the imprinted circuits persisted over time, and stimulating a single cell in the neural circuit could trigger the entire pattern.

“At least it’s very easy to build these ensembles in the cortex and also recall them later,” says Yuste, even if it remains unclear what specific functional role the neural ensembles play.

Yuste’s work is just one example of how a growing range of sophisticated optical imaging techniques are helping neuroscientists map signaling and correlate neural pathways with behavior in living organisms. From chemical agents to clarify opaque brain tissue samples to improvements in spatial and temporal resolution through light-sheet and multiphoton microscopy, neuroscientists now have a toolkit for uncovering the brain’s specialized structures and functions.

A clearer picture

Imaging neurons within the brain is not a trivial matter. Like many other tissues, the brain is made up of a host of biomolecules, particularly lipids, that scatter light as it travels through them. Depending on when and where light scattering occurs in a sample, this can prevent deep penetration of light, minimizing the excitation of fluorophores and ultimately decreasing the resolution of the detected image. Though a German researcher had done some work to make heart tissue transparent in the early 20th century, Stanford University researchers capitalized on this idea with brain tissue in 2012.

The CLARITY method was developed in the laboratory of Karl Deisseroth at Stanford University. Today, it is one of the more widely used techniques for making opaque tissues transparent. The protocol requires researchers to infuse samples with a combination of formaldehyde, acrylamide, and a thermal initiator. Monomers crosslink with the amine moieties in biomolecules, and heating prompts the hydrogel polymerization reaction. The resulting soft hydrogel structure serves as a support, making it possible to actually remove lipids, the primary molecules responsible for light scattering. This passive clearing process takes roughly 2–3 weeks for a mouse brain, according to Raju Tomer of Columbia University, who worked as a postdoctoral fellow in Deisseroth’s lab. The resulting transparent sample is a hybrid of tissue and hydrogel. Since CLARITY first emerged, a variety of tissue clearing procedures such as Scale, SeeDB, CUBIC, and LUMOS have been developed, allowing researchers to improve the optical properties of samples and acquire high-resolution images from ever-larger pieces of cortex, whole mouse brains, and even peripheral neural connections in whole mice.

With a cleared tissue sample, researchers can choose from an array of optical techniques to map tissue structure, including confocal, two-photon, or light-sheet microscopy. According to Tomer, light-sheet microscopy, which shines a plane of light into a sample and collects the emitted signal using an orthogonally arranged objective, is a natural pairing with cleared tissue. When he was in Deisseroth’s group, Tomer developed a technique that brought these ideas together: CLARITY-optimized light-sheet microscopy (COLM) (2). Whereas confocal and two-photon microscopy are both point-based methods and require more time and photons for high-resolution imaging, light-sheet microscopy shines a plane of light into a sample, and scans can be orders of magnitude faster.

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