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Heading into light-sheet microscopy at light speed: How Vanderbilt’s new neurovisualization core is removing technical barriers

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An artistic interpretation of a two-dimensional brain representation generated by José Maldonado through whole-brain imaging carried out at the Vanderbilt Neurovisualization Lab. The illustration was made by the author of the article, Kendra H. Oliver.
An artistic interpretation of a two-dimensional brain representation generated by José Maldonado through whole-brain imaging carried out at the Vanderbilt Neurovisualization Lab. The illustration was made by the author of the article, Kendra H. Oliver.

Kendra H. Oliver

José Maldonado has traveled the world over for his love of microscopes. After receiving his doctoral degree in neurobiology from UCLA and working for a company that specialized in quantitative microscopy, he became concerned about the inadequate availability of this technology in poorly served regions of the world. So, he established his own microscopy franchise in South America and Africa, where he worked to increase the sales of and the necessary training for innovative microscopy instrumentation and approaches.

Maldonado spent five years living in Brazil and traveling all around South America and Africa helping to build unique microscopes. One of the instruments that he helped to create was so massive that it could handle a tissue cross-section from an elephant’s brain. Eventually, however, he began to think that with so much of his time being spent convincing people to buy or build new microscopes, maybe he could expand his impact by helping others apply new technologies to ongoing research programs. To accomplish this goal, he thought, would require a return to academia.

Four images of mouse brains that have been made transparent and that each glow with different colors and in different patters depending on what protein is labeled on each one. From left to right, the brains have silver, orange, purple/cyan, and purple coloring.
Cleared mouse brains light up at sites of reporter protein expression, illuminating the tracts of neurons that make up distinct circuits. Whole-mouse brain projections are “stitched” together from thousands of images. Images by Joseph Luchsinger.

 

Unfortunately, there is no well-advertised mechanism for transitioning from the private sector to academia, and Maldonado traversed a difficult and convoluted path along the way. He kept running into minor issues, such as not being able to be included in someone’s grant application because he didn’t have a university affiliation. In the end, it took just the right situation and combination of events to make it work: Maldonado met Richard Simerly, professor of molecular physiology and biophysics at Vanderbilt University and the initial driver of the Vanderbilt Neurovisualization Lab.

Simerly wanted to use optical tissue clearing—making tissue transparent—and light-sheet microscopy to establish an efficient platform for visualizing complex neural circuits within the context of an intact brain. Although it was clear that such an imaging platform would be broadly valuable to the Vanderbilt neuroscience community, several of its components had to be identified and integrated. Joining the Simerly lab as a research instructor in molecular physiology and biophysics allowed Maldonado to make the career move to academia and explore the potential of light-sheet microscopy.

Light sheets ahead

Researchers using traditional microscopy to study cellular or tissue morphology slice up the organ of interest like pieces of deli meat, prepare and image them separately, and then reconstruct them digitally. This process can come with a few issues, such as the potential for damage to the tissue, the requirement of extensive labor for the preparation of many samples, and the challenge of accurately assembling the whole from the many parts.

“When you image a tissue section in a conventional microscope, it really is like looking down at the world through a straw,” Maldonado explained. “You’re looking down at one little piece, and you’re taking all these little pictures through that straw. And then you have to stitch it all together in your head.”

However, a whole new world emerges with the application of light-sheet microscopy, which allows researchers to keep the tissue intact so that they can view an entire organ without having to reconstruct structures from sections. “A particularly powerful application of single-plane illumination microscopy, a form of light-sheet technology, is to visualize the brainwide organization of neural circuits. Instead of using a knife to cut sections through the brain, we use light to create virtual slices in order to construct 3D renderings of neurons and their connections, while maintaining their in vivo orientation. It’s a potent tool for looking at whole-organ anatomy.”

The origin story

Simerly, Louise B. McGavock Chair in Molecular Physiology and Biophysics, studies how environmental factors, such as nutrition and hormones, impact the development of neural circuits that control behavior and metabolism to better understand how early events in an individual’s life influence traits like feeding and overall physiology. Goal-directed behavioral decisions—such as feeding, a primary focus in the Simerly lab—result from the neural integration of signals from the external environment with sensory information—sight, taste, and smell, for instance—that reveals the internal state to the brain. The Simerly lab explores how all of this information is conveyed to key circuit nodes responsible for goal-directed behaviors, and how the complex neural connections develop under the influence of environmental factors such as nutrition.

In particular, the lab studies the hypothalamus, a part of the brain that has a vital role in controlling many bodily functions, which has long been known for its dominant role in feeding regulation. “The neural circuits that control feeding extend from the cerebral cortex to the brainstem, so gaining a brainwide understanding of how these circuits are organized requires a new way of visualizing their architecture and development,” Simerly said.

With critical support from Larry Marnett, dean of basic sciences, Simerly and Maldonado began working together three years ago to assemble an effective platform for visualizing brain circuitry. At this point, light-sheet microscopes were available, but they were not widely adopted and resolution was somewhat limited.

“José brought a unique blend of expertise in optics, microscopy, and digital technologies to this project, as well as a fanatical pursuit of the enhancement of the signal-to-noise ratio,” Simerly said. “Working closely with members of my lab, José assembled and optimized tissue visualization and data capture technology to reveal a variety of interconnected neural systems.”

Another development was the emergence of novel tissue-clearing technologies and a new commercially available light-sheet microscope developed by Life Canvas Technologies, a Boston-based company that grew out of an academic lab. Maldonado forged a powerful collaboration with Life Canvas to accelerate the adaptation of their new tools for a whole-brain analysis platform. “This is a great example of a public-private partnership. José’s expertise and knowledge of their world was a perfect match,” Simerly said.

Immunofluorescence image of the developing nervous system of a mouse embryo. You can clearly see the spine along the top of the embryo and nerves shooting off from there.
Researchers in the lab of Richard Simerly are working to expand the reach of the VNL. Serena Sweet of the Simerly lab taught fellow graduate student Laura Geben, of the Rebecca Ihrie lab in the Department of Cell and Developmental Biology, the VNL ropes. As part of Geben’s training, they used a tissue-clearing technique called iDISCO+, combined with light-sheet fluorescence microscopy, to visualize a protein that marks the developing nervous system of a mouse embryo. Image by Laura Geben and Serena Sweet.

The new light-sheet imaging platform is beginning to bear fruit. In work that was recently published in the journal Science Translational Medicine, Michelle Bedenbaugh, a postdoctoral fellow in the Simerly lab, and Maldonado showed that the melanocortin-3 receptor is much more widely expressed in hypothalamic circuits than previously thought. MC3R is a receptor that sits on the surface of neurons and has a role in energy homeostasis. The research, conducted in collaboration with Roger Cone, director of the Life Science Institute at the University of Michigan, showed that MC3R is expressed differently in males and females and may be an important target for novel drugs to treat both anorexia and obesity.

Although powerful, these complex and costly technologies are challenging for labs to integrate into their research programs in isolation. “I am certainly not the first person to use light-sheet microscopy,” Maldonado said, but he did have to overcome a few barriers when he first began working in the field. “I found the use of the light-sheet microscope to be highly artisanal. It required a lot of attention, technical knowledge, and a lot of time to work with the machine to get a good result.” He made it his goal to guarantee that this technology was accessible to researchers to use without having to put in all of the preparatory work.

Thus, the Vanderbilt Neurovisualization Laboratory was born.

Getting the word out

Early supporters of the core were Danny Winder, professor of molecular physiology and biophysics, and lab members Joseph Luchsinger and Samuel Centanni, who worked with Maldonado and other members of the Simerly lab to identify a neuronal circuit in the brain that plays a role in the response to restraint-induced stress.

Their work, published in the journal Nature Communications, revealed the location of the circuit and outlined other connections that provide input to and receive information from it. They showed how input from the motor cortex activates the circuit before a restrained animal begins to struggle, and their results also suggest that the circuit plays a role in the affective (emotional) behavior displayed in response to the stress.

“This paper was the result of a shared interest of the Winder and Simerly labs in how forebrain circuits coordinate key aspects of motivated behavior and provided a perfect test case for the light-sheet imaging approach José developed,” explained Simerly. “The results illustrate how taking a brainwide approach to neural circuit architecture uncovers new functional targets and focuses attention on the most important pathways. This capability will be important to a wide variety of Vanderbilt research programs and documents the utility and opportunities created by the VNL,” he said, referring to the Vanderbilt Neurovisualization Lab.

A cleared mouse brain (it looks transparent and yellow) is secured to a sample holder in preparation for imaging. It's being held between a forefinger and a thumb.
A cleared mouse brain is secured to a sample holder in preparation for imaging.

Maldonado and the new VNL, often called the NeuroViz core, are hoping to expand their success further within the Vanderbilt research community. Maldonado described his drive: “If I were going to produce a product for the private sector, how would I make this as streamlined and easy to use for a customer as possible? Except that, instead of selling a product, what I am really trying to do is enable people to look beyond the pretty picture and think about quantifying things.”

To this end, Maldonado continues to integrate resources and establish partnerships across the academic and private sectors to offer a set of readily accessible analytical tools for researchers. The goal is to make it easy for someone to both generate images and do the associated quantitative analysis. But this has not been a simple task.

Everything has been customized to increase usability. Maldonado scoured GitHub—a cloud-based repository for software development projects—for existing code to use for automatic quantification. “There were a couple of them that were failures,” Maldonado explained. Over the last few years, Maldonado has been augmenting the existing system, using the expertise of commercial partners and implementing various tools to get his turnkey quantitative software set up.

The mouse brain from the previous image is now contained in a liquid-filled chamber. You can see green laser light going through the middle of it and making it glow green. The image is taken at an angle (on purpose) so the photo looks noticeably crooked.
Laser light is formed into a sheet and passed through the sample, which is now immersed in a liquid-filled chamber.

“The Nature Communications paper demonstrates the power of what we’ve been able to establish: the ability to image cleared tissue and quantify cell numbers based on anatomical brain regions through the application of unbiased computational methods,” he said. Even though this sort of quantitation and mapping has been published, it was not previously possible to do that sort of analysis easily or to that scale.

The Vanderbilt Neurovisualization Lab represents a fine-tuning of the application of light-sheet microscopy for neuroscience, and Maldonado now feels that he knows how to make an impact as the core director.

A zoomed-out view of the same liquid chamber, which you can now see is part of a microscope. A specialized lens sits on top of the liquid, looking down toward the brain. The laser light is now blue. A hand can be seen adjusting a setting on the microscope.
A specialized lens is carefully calibrated into place for the three-dimensional imaging of the sample.

“A lot of the things that I am faced with in academia mirror the kind of things that I helped people do in the private sector,” Maldonado said. “Basically, I am trying to make microscopy technology a little bit more accessible.”

However, in contrast to being in the private sector, Maldonado now gets to drive every aspect of the work that he does and distribute his expertise across multiple research programs. “When you see a scientist look at one of their images for the first time, that’s the payoff,” Maldonado said. “When they see the brain this way, the look on people’s faces is the most impactful part for me. So, right now, I want to get this in front of as many eyes as possible. But more importantly, I want to get the point across that this is something that is accessible to every researcher’s lab.”

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