Scientists are gaining new insight into muscular dystrophy using a form of high-resolution fluorescence microscopy.

A team from the University of Southern California developed the technique, which allows the viewing of individual molecules in living tissue. Complementation-activated light microscopy (CALM) offers imaging resolutions an order of magnitude finer than conventional optical microscopy, and a better look at the behavior of biomolecules at the nanometer scale.

“Now, for the first time, we can explore the basic principles of homeostatic controls and the molecular basis of diseases at the nanometer scale directly in intact animal models,” said Dr. Fabien Pinaud, an assistant professor at the USC Dornsife College of Letters, Arts and Sciences.

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The new single-molecule imaging technology offers new insights into the role of dystrophin proteins for muscle function in worm models of muscular dystrophy. Courtesy of Dr. Fabien Pinaud.

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Researchers have used CALM to look at dystrophin, a key structural protein of muscle cells, in live C. elegans worm models to study Duchenne muscular dystrophy, which is the most severe and common form of this degenerative disease.

The researchers used split green fluorescent proteins which, when united, emit light that CALM can detect with high accuracy and imaging precision.

Tissue-specific, single-molecule tracking was achieved in vivo with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms, the researchers wrote in the study. They noted that conventional optical microscopy of living tissues typically achieves 200-nm resolution.

The results show that dystrophin is responsible for regulating tiny molecular fluctuations in calcium channels while muscles are in use. This suggests that a lack of functional dystrophin alters the dynamics of ion channels, “helping to cause the defective mechanical responses and the calcium imbalance that impair normal muscle activity in patients with muscular dystrophy,” the researchers wrote.

“There are trillions of proteins at work on an infinitely small scale at every moment in an animal’s body,” Pinaud said. “The ability to detect individual protein copies in their native tissue environment allows us to reveal their functional organization and their nanoscale molecular behaviors despite this astronomical complexity.”

The researchers next plan to engineer other colors of split fluorescent proteins to image the dynamics of individual ion channels at neuromuscular synapses within live worms.

The research was published in Nature Communications (doi: 10.1038/ncomms5974). 

For more information, visit www.usc.edu.

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