By combining three types of microscopic imaging techniques that are ordinarily used individually, a way has been developed to analyze living tissue and learn more about the molecular mechanisms of multiple sclerosis, information that could help lead to earlier detection and new treatments. The combined imaging method is enabling the researchers to study how multiple sclerosis causes an overproduction of "astroglial filaments," which form bundles between critical nerve fibers and interfere with proper spinal cord functioning. The technique also promises to yield new information about how the disease degrades the myelin sheath, which insulates nerve fibers and enables them to properly conduct impulses in the spinal cord, brain and in the "peripheral nervous system" throughout the body, said Ji-Xin Cheng, an assistant professor in Purdue University's Weldon School of Biomedical Engineering and Department of Chemistry. Purdue University researchers have shown how to combine three microscopic imaging techniques in one platform to analyze living tissues. This photo was taken with two of the techniques combined, enabling researchers to conduct more specific and precise molecular analyses for research regarding multiple sclerosis and other conditions. The myelin sheath image, in red, was taken via coherent anti-Stokes Raman scattering, while the astroglial filaments image, in green, was achieved through sum frequency generation. (Image courtesy Weldon School of Biomedical Engineering, Purdue University) The three imaging techniques -- called sum frequency generation, two-photon excitation fluorescence and coherent anti-Stokes Raman scattering -- ordinarily are used alone. Purdue researchers have developed a way to combine all three methods in the same platform, promising to reveal new details about the spinal cord and myelin sheath, Cheng said. "Combining these three methods allows us to conduct more specific and precise molecular analyses," he said. "Ultimately, this work paves the way toward studying the degradation of the myelin sheath as a result of multiple sclerosis and analyzing living tissue to study the mechanisms of disease." Multiple sclerosis, or MS, affects more than 350,000 people in the US and 2 million worldwide. The findings will be detailed in a paper appearing in May in the Biophysical Journal and is currently online. The paper was authored by biomedical engineering doctoral student Yan Fu and postdoctoral research associate Haifeng Wang; Riyi Shi, an associate professor of basic medical science in Purdue's School of Veterinary Medicine and also an associate professor of biomedical engineering; and Cheng. Shi, a member of Purdue's Center for Paralysis Research, specializes in spinal cord and brain trauma and chronic neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and MS. "We are using a unique and powerful combination of technologies to uncover the mechanisms of multiple sclerosis," Shi said. "We hope to one day establish an effective intervention to not only slow down, but even possibly reverse the development of this disease, which will potentially have profound economic and social impacts on this nation and the world." Because the imaging techniques work without using dyes to "label" cells and structures, they can be used to study living tissues, representing a major advantage over conventional microscopic imaging technologies. Raman microscopy, an imaging technique invented more than three decades ago, cannot be used effectively to study living tissue because the extremely weak "Raman scattering" signals require hours to yield an image, whereas coherent anti-Stokes Raman scattering, or CARS, overcomes this limitation, Cheng said. "CARS microscopy permits label-free imaging of specific molecules with a speed of one frame per second or even faster," he said. CARS imaging takes advantage of the fact that molecules vibrate at specific frequencies. In a CARS microscope, two laser beams are overlapped to produce a single beam having a new frequency representing the difference between the original two beams. This new frequency then drives specific molecules to vibrate together "in phase," amplifying the Raman signals from those molecules. "It's like pushing someone on a swing," Cheng said. "If you push in synch with the upswing, the swing will go higher. That's the same as being in phase." Sum frequency generation imaging does just the opposite, adding the frequencies of the two original beams, producing a new signal with a frequency that is the sum of the original beams. The third imaging method, two-photon excitation fluorescence, provides higher contrast and brighter images than conventional fluorescent imaging techniques. Photons are the individual particles that make up light; in two-photon excitation fluorescence, two photons are used to illuminate a target. The researchers have used the imaging methods to observe living spinal tissue extracted from guinea pigs. The technique of extracting the tissue and then keeping it alive long enough to analyze was developed in Shi's lab. Conventional microscopic imaging techniques require samples to be labeled with dyes, killing the tissues in the process. Being able to analyze living tissue could allow researchers to determine the molecular mechanisms responsible for multiple sclerosis and other conditions, Cheng said. The myelin sheath is made of molecules called lipids, which consist of a head and tail segment. The new findings show that images can reveal whether the orientation of the chemical bonds in these lipid molecules is "scrambled," when the myelin sheath is unhealthy and degraded from disease, Cheng said. "We can see the myelin sheath with CARS, and that's great, but it's not enough," Cheng said. "We also want to study other molecules and see the interactions between cells." Researchers simultaneously took images of astroglial filaments, critical components of structures called astrocyte processes, which provide structural support for the nerve fibers in a spinal column. The sum frequency generation imaging reveals details about the astroglial filaments. Two-photon excitation fluorescence reveals information about the influx of calcium into cells, which causes damage to nerve fibers. "So, having all three of these imaging methods in the same platform enables us to study many elements of the disease process simultaneously in the same tissue samples," Cheng said. Researchers hope one day to use the combined approach to design a "minimally invasive" system for diagnosing patients in the hospital or doctor's office, he said. "There are two directions of this research," Cheng said. "One is to study the mechanisms of disease, and that should form the foundation for designing new treatments. The other is to keep pushing the technology to make it less and less invasive, which will help in the early detection of multiple sclerosis." The astroglial filaments also are involved in producing scar tissues following trauma injuries to the central nervous system, so a better understanding of their workings could lead to new treatments for repairing damage caused in accidents. The research was supported with funds from the National Science Foundation and the State of Indiana. Future work by the same researchers to study MS in rats will be supported by a new three-year, $1 million grant from the National Institutes of Health's National Institute of Biomedical Imaging and Bioengineering. For more information, visit: www.purdue.edu