Researchers have developed a way to use three types of microscopic imaging techniques simultaneously 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.
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 affects more than 350,000 people in the United States and 2 million worldwide.
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 multiple sclerosis.
"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 technique, two-photon excitation fluorescence, provides higher contrast and brighter images than conventional fluorescent imaging methods. Photons are the individual particles that make up light. In two-photon excitation fluorescence, two photons are used to illuminate a target.