Up Close and Personal

From the Winter 2018 edition of Vanderbilt Medicine Magazine

High-spatial resolution mass spectroscopy reveals in bold colors the distribution and concentration of different lipids based on their molecular weight in a rat brain. Image courtesy of Richard Caprioli, PhD.

 

Recent advances in imaging technology are enabling scientists to “see” how molecules, cells and tissues are put together — and work together. These unprecedented views of life down to the minutest level are yielding radical new insights into the causes, treatment and prevention of disease.

At Vanderbilt University and Vanderbilt University Medical Center (VUMC), a long-term commitment to imaging science has nurtured an expertise and technological prowess matched by only a handful of the nation’s leading universities and academic medical centers.

Researchers in the Center for Molecular Probes and Department of Chemistry, for example, are developing new radiopharmaceuticals — tracers — used in positron emission tomography (PET) to improve detection of cancer and other diseases. “Our efforts literally bridge molecules to man,” said center director Charles Manning, PhD.

Across the Vanderbilt campus, researchers are applying high- resolution light microscopy and cryo-electron microscopy to generate amazingly detailed pictures of cell structures and the complex molecular “machines” that do the cell’s work. At the brink: image fusion, a brilliant combination of existing techniques that has the potential to transform medical practice.

“Seeing leads to understanding,” said Lawrence Marnett, PhD, dean of Basic Sciences and the Mary Geddes Stahlman Chair in Cancer Research in the Vanderbilt University School of Medicine.

Thanks to the latest imaging technology, “Vanderbilt scientists can determine the three-dimensional structures of molecular machines then watch them operate in intact cells, tissues or people,” Marnett said. “This helps us understand how they function in normal cellular physiology and what goes wrong in disease.

“Each of our imaging technologies operates at the state-of-the-art,” he said. “They have been implemented by Vanderbilt scientists who are defining the frontiers of their fields. The breadth and depth of our capabilities are awesome. This is a tremendous resource for our researchers, clinicians and patients.”

 

Precision medicine

The development of Vanderbilt’s imaging infrastructure owes much to Trans-Institutional Programs (TIPs), a series of far-sighted grants that support cutting-edge research and infrastructure development. Part of the University’s $50 million strategic investment in interdisciplinary research, the annual TIPs initiative was launched in 2014.

A 2015 TIPs grant is aiding development of high-field magnetic resonance imaging (MRI) for advanced neuroimaging at the Vanderbilt University Institute for Imaging Science. A 2016 grant is supporting the search for novel antibodies and the design of next-generation vaccines by the Vanderbilt Vaccine Center and Center for Structural Biology.

Another 2015 grant is supporting development of small, microfluidic-based reactors that enable “on-demand” production of inexpensive, single-patient doses of new PET imaging agents, a radical advance that is pushing forward the frontier of molecular imaging in clinical settings.

“We have about a dozen investigational radiopharmaceuticals in clinical trials at Vanderbilt,” said Manning, the Ingram Professor of Cancer Research.

One innovative PET tracer called FSPG detects tumors by their increased rate of glutamine metabolism. Using a “theranostics” approach, Manning and his colleagues are testing a drug in animals that blocks glutamine uptake. By labeling the drug with an imaging isotope, they can see where it goes.

The goal is precision medicine — matching patients with the right therapy and monitoring outcomes with PET. “If used appropriately,” Manning said, “PET can reduce the cost of healthcare because we can make better decisions about the diagnosis and the treatment the patient should get.”

 

Machines of life

A second revolution brewing at Vanderbilt is the application and refinement of cryo-electron microscopy (cryo-EM). The 2017 Nobel Prize in Chemistry was awarded to the developers of this ground-breaking technique, which enables researchers to directly visualize biomolecules and molecular complexes.

For decades, X-ray crystallography has been the gold standard for visualizing the structures of proteins at the atomic level. But it’s not perfect. Some molecules and complexes will not crystallize and the crystalline environment sometimes induces artifacts.

In the past few years, cryo-EM solved these problems and set off a revolution in the field. Single molecules and molecular complexes can be directly observed without any need for crystallization.

This confocal microscope image shows a Tuft cell (green) in the intestinal epithelium. Tuft cells are rare host defense cells that respond to infection in the gut. Image courtesy of Matthew Tyska, PhD.

That’s important, said Walter Chazin, PhD, Chancellor’s Professor of Medicine and director of the Center for Structural Biology, because it’s these protein complexes, what he calls “machines of life,” that are key to understanding and developing more effective treatments for diseases like cancer.

“What’s happened in cryo-EM in the last five years has just been a stunning development in science,” added Charles Sanders, PhD, the Aileen M. Lange and Annie Mary Lyle Professor of Cardiovascular

Research and associate dean for Research. “And we’re just getting started on this.”

Already the next-generation cryo-EM is on the market, and Vanderbilt is in line to get one, thanks to a TIPs grant awarded in 2017. Called the Titan Krios, the instrument sells for more than $6 million. It will be delivered to Vanderbilt in the fall.

“We really have to have this technology here,” said Borden Lacy, PhD, the Edward and Nancy Fody Professor of Pathology who is studying a bacterial toxin associated with diarrheal illness. “We have an excellent instrument, but the Titan Krios is bringing (image) stability and automation we don’t have.”

 

Molecular mapping

A third imaging revolution at Vanderbilt has to do with super-resolution light microscopy.

Before 2011 light microscopes used by Vanderbilt scientists were limited to a resolution of about 200 nanometers. That’s not sharp enough to pick up individual molecules within cells, which might be only a few nanometers across.

Thanks to a computational advance called STORM (Stochastic Optical Reconstruction Microscopy), scientists now can calculate the size and location of molecules in cells and tissues.

“You can build maps of where all the molecules are,” said Matthew Tyska, PhD, scientific director of the Cell Imaging Shared Resource (CISR), the Cornelius Vanderbilt Chair and professor of Cell and Developmental Biology.

STORM and another technique called SIM (Structured Illumination) are offered through the Nikon Center of Excellence for live-cell imaging at Vanderbilt, one of six in the country.

The center opened in 2016 with financial support from the medical school’s Department of Cell and Developmental Biology and the office of the Dean of Basic Science and with technical support from Nikon.

A newer method, called light-sheet microscopy, uses a thin plane of light to rapidly “section” extremely large biological samples. The resulting three-dimensional information can then be reconstructed in a computer.

In this positron emission tomography (PET) scan fluoro-thymidine (FLT) tracer detects enhanced DNA synthesis in rapidly dividing rectal cancer cells (spot at bottom, middle). Rapid DNA synthesis in healthy bone marrow and normal uptake of the tracer by the liver and clearance by the kidneys can be seen as well. Image courtesy of Charles Manning, PhD.

Installed in December 2017, Vanderbilt’s $340,000 LaVision Ultramicroscope II “greatly expands our ability to image fluorescent signals in large volumes of brain and other tissues,” said Richard Simerly, PhD, the Louise B. McGavock Professor in the medical school’s Department of Molecular Physiology and Biophysics.

Simerly was recruited in 2016 from the University of Southern California and is developing a neuro-visualization core.

Researchers in the Schools of Medicine and Engineering and in the College of Arts and Science are now working on the next technological advance — lattice light-sheet microscopy. It will enable scientists to perform long-term, high-resolution imaging over time down to the molecular scale — minutes-long “movies of life,” Tyska said.

A TIPs grant awarded in 2017 is supporting the effort to build the new microscope, which is not yet commercially available.

Anita Mahadevan-Jansen, PhD, the Orrin H. Ingram Professor of Biomedical Engineering, is the grant’s principal investigator (PI).  Tyska and Shane Hutton, PhD, chair of the Department of Physics and Astronomy in the College of Arts and Science, are co-PIs.

 

Disease signatures

Despite all the recent progress, clinical imaging techniques including MRI cannot “see” beyond the millimeter level. But it’s at the micron to molecular scale where disease occurs. “Somehow we’re going to have to figure out a way to close that gap,” Simerly said.

That’s where a fourth research revolution at Vanderbilt comes in: imaging mass spectrometry (IMS) and image “fusion.”

IMS, developed at Vanderbilt in the late 1990s by Richard Caprioli, PhD, and his colleagues, is essentially a “molecular microscope” that can measure the distribution, spatial rearrangement and alteration in expression levels of proteins, lipids and other biological molecules.

In 2015, Caprioli’s team reported the first “image fusion” of mass spectrometry and microscopy, a technological tour de force that allows scientists to see the molecular make-up of tissues in bright-field microscopic resolution.

“We’re now coming up with (molecular) signatures for disease,” said Caprioli, the Stanford Moore Professor of Biochemistry and director of the Mass Spectrometry Research Center. The technique can identify cells that look normal under the microscope but which already are transforming into cancer.

Caprioli predicts image fusion will have a huge impact on anatomic pathology and surgery. In removing a kidney tumor, where should the surgical margin be? In other words, how much tissue should be removed to minimize the chance for recurrence? Image fusion can help.

Timothy Cover, MD, professor of Medicine and of Pathology, Microbiology and Immunology, who is studying bacterial toxins, is looking forward to adding image fusion to his armamentarium of research tools.

“I think all of these things will be complementary,” he predicted. “One of the things Vanderbilt is doing very well is staying competitive in multiple imaging areas.”

Simerly agreed.

“We have some of the best cell imagers in the world here,” he said, “and we are also strong in whole brain imaging. We are well positioned to narrow the gap and improve the predictive value of clinical imaging.

“I think it’ll definitely happen,” Simerly said. “The question is when.”