With New Imaging TechniqueResearchers are using a triad of microscopy imaging approaches to delve into the workings of MS.
Researchers at Purdue Univ., West Lafayette, Ind., have devised a way to use three types of microscopy imaging techniques concurrently to analyze living tissue and discover more about the molecular mechanisms of multiple sclerosis (MS).
MS is a chronic, inflammatory disease which affects the body’s central nervous system. The disease causes a gradual destruction of the myelin sheath, a fatty layer that surrounds and protects nerve fibers and enables them to carry electrical signals from the brain. The name multiple sclerosis refers to the multiple scars (or scleroses) on the body’s myelin sheaths.
This scarring causes symptoms which vary widely depending upon which signals are interrupted, including visual problems, muscle weakness, difficulties with coordination and speech, and pain. Currently, more than 2 million people worldwide suffer from MS.
Three ways are better than one
The trio of nonlinear optical imaging techniques being used by the Purdue researchers—coherent anti-Stokes Raman scattering (CARS), two-photon-excitation fluorescence (TPEF), and sum frequency generation (SFG)—are each normally used alone. But used together, they allow simultaneous visualization of different structures in a complex biological system.
Raman microscopy, an imaging technique invented more than 30 years ago, cannot be used effectively to study living tissue because the extremely weak Raman scattering signals require hours to yield an image. CARS overcomes this limitation. Ji-Xin Cheng, assistant professor in Purdue’s Weldon School of Biomedical Engineering and Dept. of Chemistry explains that “CARS microscopy permits label-free imaging of specific molecules with a speed of one frame/sec or even faster.” Cheng says 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 says. “If you push in synch with the upswing, the swing will go higher. That’s the same as being in phase.”
SFG 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.
TPEF, the third imaging technique, provides higher contrast and brighter images than conventional fluorescent imaging methods. In two-photon excitation fluorescence, two photons are used to illuminate a target.
This unique method of combining imaging techniques enables the researchers to study how MS causes an overproduction of astroglial filaments, which form bundles between critical nerve fibers and interfere with proper spinal cord functioning. Astroglial filaments also are involved in producing scar tissue following traumatic injuries to the central nervous system, therefore a better understanding of their workings could lead to new treatments for repairing damage caused in accidents as well.
“The technique also promises to yield new information about how the disease degrades the myelin sheath”, says Cheng. 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, according to Cheng, “scrambled,” when the myelin sheath is unhealthy and degraded from disease.
Viewing it live
An added benefit to using this triad of imaging techniques is that no dyes are needed. This represents a major advantage over conventional microscopic imaging techniques as living tissues can be studied. Through this study, researchers hope to one day reverse the development of MS.
“There are two directions of this research,” Cheng says. “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 MS.”
So hopeful are the researchers about their study, that Riyi Shi, associate professor of basic medical science in Purdue’s School of Veterinary Medicine and associate professor of biomedical engineering says they hope to one day “establish an effective intervention to not only slow down, but even possibly reverse the development of MS, which will potentially have profound economic and social impacts on this nation and the world.”
This research was supported with funds from the National Science Foundation, Arlington, Va., 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, Bethesda, Md.