Biology Made Simpler With "Clear" Tissues

In general, our knowledge of biology—and much of science in general—is limited by our ability to actually see things. Researchers who study developmental problems and disease, in particular, are often limited by their inability to look inside an organism to figure out exactly what went wrong and when.

Now, thanks to techniques developed at Caltech, scientists can see through tissues, organs, and even an entire body. The techniques offer new insight into the cell-by-cell makeup of organisms—and the promise of novel diagnostic medical applications.

"Large volumes of tissue are not optically transparent—you can't see through them," says Viviana Gradinaru (BS '05), an assistant professor of biology at Caltech and the principal investigator whose team has developed the new techniques, which are explained in a paper appearing in the journal Cell. Lipids throughout cells provide structural support, but they also prevent light from passing through the cells. "So, if we need to see individual cells within a large volume of tissue"—within a mouse kidney, for example, or a human tumor biopsy—"we have to slice the tissue very thin, separately image each slice with a microscope, and put all of the images back together with a computer. It's a very time-consuming process and it is error prone, especially if you look to map long axons or sparse cell populations such as stem cells or tumor cells," she says.

The researchers came up with a way to circumvent this long process by making an organism's entire body clear, so that it can be peered through—in 3-D—using standard optical methods such as confocal microscopy.

The new approach builds off a technique known as CLARITY that was previously developed by Gradinaru and her collaborators to create a transparent whole-brain specimen. With the CLARITY method, a rodent brain is infused with a solution of lipid-dissolving detergents and hydrogel—a water-based polymer gel that provides structural support—thus "clearing" the tissue but leaving its three-dimensional architecture intact for study.

The refined technique optimizes the CLARITY concept so that it can be used to clear other organs besides the brain, and even whole organisms. By making clever use of an organism's own network of blood vessels, Gradinaru and her colleagues—including scientific researcher Bin Yang and postdoctoral scholar Jennifer Treweek, coauthors on the paper—can quickly deliver the lipid-dissolving hydrogel and chemical solution throughout the body.

Gradinaru and her colleagues have dubbed this new technique PARS, or perfusion-assisted agent release in situ.

Once an organ or whole body has been made transparent, standard microscopy techniques can be used to easily look through a thick mass of tissue to view single cells that are genetically marked with fluorescent proteins. Even without such genetically introduced fluorescent proteins, however, the PARS technique can be used to deliver stains and dyes to individual cell types of interest. When whole-body clearing is not necessary the method works just as well on individual organs by using a technique called PACT, short for passive clarity technique.

To find out if stripping the lipids from cells also removes other potential molecules of interest—such as proteins, DNA, and RNA—Gradinaru and her team collaborated with Long Cai, an assistant professor of chemistry at Caltech, and his lab. The two groups found that strands of RNA are indeed still present and can be detected with single-molecule resolution in the cells of the transparent organisms.

The Cell paper focuses on the use of PACT and PARS as research tools for studying disease and development in research organisms. However, Gradinaru and her UCLA collaborator Rajan Kulkarni, have already found a diagnostic medical application for the methods. Using the techniques on a biopsy from a human skin tumor, the researchers were able to view the distribution of individual tumor cells within a tissue mass. In the future, Gradinaru says, the methods could be used in the clinic for the rapid detection of cancer cells in biopsy samples.

The ability to make an entire organism transparent while retaining its structural and genetic integrity has broad-ranging applications, Gradinaru says. For example, the neurons of the peripheral nervous system could be mapped throughout a whole body, as could the distribution of viruses, such as HIV, in an animal model.

Gradinaru also leads Caltech's Beckman Institute BIONIC center for optogenetics and tissue clearing and plans to offer training sessions to researchers interested in learning how to use PACT and PARS in their own labs.

"I think these new techniques are very practical for many fields in biology," she says. "When you can just look through an organism for the exact cells or fine axons you want to see—without slicing and realigning individual sections—it frees up the time of the researcher. That means there is more time to the answer big questions, rather than spending time on menial jobs."

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Corals Provide Clues for Climate Change Research

Just as growth rings can offer insight into climate changes occurring during the lifespan of a tree, corals have much to tell about changes in the ocean. At Caltech, climate scientists Jess F. Adkins and Nivedita Thiagarajan use manned submersibles, like Alvin operated by the Woods Hole Oceanographic Institution, to dive thousands of meters below the surface to collect these specimens—and to shed new light on the connection between variance in carbon dioxide (CO2) levels in the deep ocean and historical glacial cycles.

A paper describing the research appears in the July 3 issue of Nature.

It has long been known that ice sheets wax and wane as the concentration of CO2 decreases and increases in the atmosphere. Adkins and his team believe that the deep ocean—which stores 60 times more inorganic sources of carbon than is found in the atmosphere—must play a vital role in this variance.

To investigate this, the researchers analyzed the calcium carbonate skeletons of corals collected from deep in the North Atlantic Ocean. The corals were built up from 11,000–18,000 years ago out of CO2 dissolved in the ocean.

"We used a new technique that has been developed at Caltech, called clumped isotope thermometry, to determine what the temperature of the ocean was in the location where the coral grew," says Thiagarajan, the Dreyfus Postdoctoral Scholar in Geochemistry at Caltech and lead author of the paper. "We also used radiocarbon dating and uranium-series dating to estimate the deep-ocean ventilation rate during this time period." 

The researchers found that the deep ocean started warming before the start of a rapid climate change event about 14,600 years ago in which the last glacial period—or most recent time period when ice sheets covered a large portion of Earth—was in the final stages of transitioning to the current interglacial period.

"We found that a warm-water-under-cold-water scenario developed around 800 years before the largest signal of warming in the Greenland ice cores, called the 'Bølling–Allerød,'" explains Adkins. "CO2 had already been rising in the atmosphere by this time, but we see the deep-ocean reorganization brought on by the potential energy release to be the pivot point for the system to switch from a glacial state, where the deep ocean can hold onto CO2, and an interglacial state, where it lets out CO2."  

"Studying Earth's climate in the past helps us understand how different parts of the climate system interact with each other," says Thiagarajan. "Figuring out these underlying mechanisms will help us predict how climate will change in the future." 

Additional authors on the Nature paper, "Abrupt pre-Bølling–Allerød warming and circulation changes in the deep ocean," are geochemist John M. Eiler and graduate student Adam V. Subhas from Caltech, and John R. Southon from UC Irvine. 

Katie Neith
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BBE Hosts Symposium to Honor Patterson

On June 30, the Division of Biology and Biological Engineering sponsored a neuroimmunology symposium dedicated to the life and career of Paul Patterson, the late Anne P. and Benjamin F. Biaggini Professor of Biological Sciences, Emeritus, who died on June 25. The symposium, titled "From the Brain to the Body and Back: A Celebration of Paul Patterson's Life in Science," highlighted Patterson's work—as well how the fundamental findings from his research influenced the work of his former students, postdocs, and colleagues.

Caltech speakers at the symposium included David Anderson, Seymour Benzer Professor of Biology; Elaine Hsiao (PhD '13), senior research fellow; and Sarkis Mazmanian, Luis B. and Nelly Soux Professor of Microbiology.

Anderson spoke about Patterson's work in stem-cell lineages and neuropoesis—the process through which neural stem cells differentiate into mature neurons—and how this work sparked his interest in neural stem cells and influenced his decision to join the Caltech faculty in 1986.

Hsiao, the most recent graduate-student alumnus from Patterson's research group, spoke about how Patterson was a dedicated mentor, expecting scientific rigor from members of his group, but also encouraging them to follow their curiosity. Not only did Patterson dedicate himself to understanding complex neurodevelopmental disorders such as schizophrenia and autism, Hsiao said, but he included in his concern the people affected by these disorders, directly engaging and communicating with the schizophrenia and autism communities through his blog.

Mazmanian, Patterson's longtime collaborator, reflected on Patterson's breadth of work over the course of a research career that spanned disciplines from membrane biochemistry to stem-cell differentiation to disease. A recent collaboration between Mazmanian and Patterson revealed that specific probiotic therapies might be a treatment option for the behavioral symptoms of autism. Before Patterson's death, the two were pursuing a clinical trial to test the therapy in humans; Mazmanian said that he intends to continue this pursuit in Patterson's honor.

Several other former Caltech graduate students and research fellows who worked in Patterson's laboratory also gave presentations on their research and memories, including former graduate student Mahendra Rao (PhD '91) of the New York Stem Cell Foundation Research Institute; former research fellows Zaven Kaprielian of Amgen and Hiroyuki Nawa of the Niigata University Brain Research Institute; and Hiroshi Ueda of Nagasaki University, a former visiting scientist at Caltech.

For a full list of speakers and more information about the symposium, please visit the neuroimmunology symposium webpage.

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Noted Neuroscientist Paul Patterson Dies

Paul H. Patterson, the Anne P. and Benjamin F. Biaggini Professor of Biological Sciences, Emeritus, at Caltech, and a neuroscientist and developmental biologist who created novel behavioral models of schizophrenia and autism in mice, died on Wednesday, June 25. He was 70 years old.

Patterson's research focused on interactions between the nervous and immune systems—a connection that was not universally acknowledged in the early days of neuroscience. "Professor Patterson was a pioneer and iconoclast who was not afraid to work outside the scientific mainstream, and who consequently made a number of important and seminal contributions that opened up entire fields of research," says David Anderson, Seymour Benzer Professor of Biology at Caltech and Patterson's longtime colleague.

One such contribution came early in his career when he began a postdoctoral fellowship, which eventually became a faculty position, in the department of neurobiology at Harvard Medical School. There, he developed a laboratory technique for growing peripheral neurons—nerve cells that are not part of the brain or spinal cord—in a culture. Using this new technique, he found that mature peripheral neurons could change their identity in response to environmental influences, demonstrating that each cell's identity is not genetically determined and absolute, a fundamental discovery in neuroscience.

In 1983, after more than a decade at Harvard, Patterson joined the faculty at Caltech, where his uncle Clair Patterson was a longtime faculty member in the division of geological and planetary sciences. At Caltech, Paul Patterson built upon his previous work at Harvard, seeking to characterize the puzzling factor that caused those mature neurons to change their identity. In 1989, genetic sequencing revealed that the factor was a cytokine, or cell-signaling molecule, called leukemia inhibitory factor (LIF), which had previously been identified based on its immunological function.

This finding influenced Patterson's initial involvement in the field of neuroimmunology. His newfound interest in working at the intersection of neuroscience and immunology led to his becoming increasingly curious about the biology of inflammation and its impact on the developing brain and on behavior.

As part of this work, he became intrigued by epidemiological studies that had linked a severe viral or bacterial infection during pregnancy with the increased risk of a woman giving birth to a child with a neurodevelopmental disorder such as schizophrenia or autism. Patterson and his coworkers reproduced this human effect in mice using a viral mimic that triggers an infection-like immune response in the mother, producing in the offspring the core behavioral symptoms associated with autism and schizophrenia.

Since its development in 2007, Patterson's mouse model has been used to study the environmental factors that influence the symptoms of human neurodevelopmental disorders and has increased awareness of the importance of those influences. Recently, the model informed a possible new therapy to treat schizophrenia-associated hallucinations.

In another recent study, Patterson and colleagues demonstrated that the gut microbiome, the diverse collection of bacteria that reside in the intestine, regulates behaviors in a mouse model of autism, and that probiotic treatment leads to improvements in behavioral deficits. These studies—a collaboration with Caltech microbiologist Sarkis Mazmanian—suggest that neurodevelopmental disorders with strong environmental influences may be ameliorated with microbial therapies.

"It will be a breakthrough to link the possible role of gut bacteria to autism, and I believe it gave him an explanation of how environmental components, about which he had speculated for years, contribute to the disease process—and it's sad that he's not going to be here to see what will happen over the next five to ten years," says Mazmanian, Luis B. and Nelly Soux Professor of Microbiology. "I think there will be increasing acceptance in the future for the perspectives that Paul has had for over two decades."

Professor Patterson also contributed to the understanding and treatment of Huntington's disease, a devastating hereditary neurological disorder. He served on the scientific advisory board of the Hereditary Disease Foundation since 1991, and with support from this foundation he carried out work that spanned more than a decade, applying his knowledge of monoclonal antibodies to develop new therapies for this incurable disease.

Patterson's dedication to science continued even after he began his battle with a brain tumor in late 2013. He attended lab meetings with his research group until he could no longer be moved in a wheelchair—and at that point, lab meetings were moved to his home to accommodate his attendance.

"I think the biggest compliment you can give him is that he was a true scientist; he did everything with respect to the scientific process," Mazmanian says. "When he was first diagnosed with glioblastoma—before the surgery, radiation, and chemo—he called both of our labs together for a meeting, since we work so closely. He then gave a lab meeting about his own disease. He showed an MRI of his own tumor, he showed data from papers he had read about his disease, and he talked about clinical trials that were going on. It was his way of communication. It's the way he approached everything: as a scientist."

In addition to the medical and therapeutic possibilities of his own work, Patterson thought that Caltech graduate students should also have an opportunity to explore their work in a clinical setting. He was instrumental in developing the Institute's MD/PhD joint degree program—a collaboration that allows graduate students to combine their Caltech research experience with medical education at UCLA or USC.

"Paul showed creativity both in curriculum development, in student mentoring, and in bringing the Caltech faculty together to support a program, which was in collaboration with another major institution," says Richard Bergman, director of the Cedars-Sinai Diabetes and Obesity Research Institute, who helped Patterson form the initial collaboration with USC. "His contributions in this regard educated several generations of students who, today, continue to make important contributions to medical science. This was a great legacy of Professor Patterson."

Patterson was also an advocate for students both within and outside of academia, says Athena Castro, executive director of the Caltech Y. Patterson and his wife, Carolyn, were both members of the Caltech Y—an external organization that aims to broaden the horizons of Caltech students and increase their social awareness. "He cared deeply about students and their well-being, and also took his role as mentor very seriously. He made a positive impact on people's lives in many ways," she says. "Those of us involved with him at the Caltech Y will remember him for his genuine concern for and kindness toward others, his passion for his work, and his spirit."

Born in Chicago in 1943, Patterson stayed in the Midwest for college, graduating with a bachelor's degree from Grinnell College in Iowa in 1965. From there, he moved east for graduate school at Johns Hopkins University, earning his doctorate under advisor William Lennarz in 1970.

Patterson served as the executive officer for neurobiology at Caltech from 1989 to 2000. He was also the recipient of numerous awards and honors and was a fellow of the American Association for the Advancement of Science.

He is survived by his wife, Carolyn, and his son, Paul.

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