New microscopy technique images living single cells

A new microscopy technology has allowed scientists to paint a target in a living subject and watch how it works with unprecedented sensitivity and precision. Dubbed Complementation Activated Light Microscopy (CALM), the technology allows imaging resolutions that are an order of magnitude finer than conventional optical microscopy, providing new insights into the behaviour of biomolecules at the nanometre scale. Researchers from the University of Southern California used the technique to study dystrophin, a key structural protein of muscles in Caenorhabditis elegans which are used to model Duchenne muscular dystrophy. CALM works by splitting a green fluorescent protein from jellyfish in two fragments that fit together like a puzzle. One fragment is engineered to be expressed in animal test subjects while the other is injected into the animal’s circulatory system. When the two fragments meet, they unite and emit fluorescent light that can be detected at precisions of around 20nm. “Now for the first time, we can explore the basic principles of homeostatic controls and the molecular basis of diseases at the nanometre scale directly in intact animal models,” said Fabien Pinaud, assistant professor and lead researcher on the project. Results published in Nature Communications showed dystrophin is responsible for regulating tiny molecular fluctuations in calcium channels while muscles are in use. This suggests a lack of functional dystrophin alters the dynamics of ion channels, causing defective mechanical responses and calcium imbalance that impair normal muscle activity in patients with muscular dystrophy. "There are trillions of proteins at work on an infinitely small scale at every moment in an animal's body. The ability to detect individual protein copies in their native tissue environment allows us to reveal their functional organisation and their nanoscale molecular behaviours despite this astronomical complexity," Pinaud said. Researchers will focus on engineering other colours of split-fluorescent proteins to image the dynamics of individual ion channels at neuromuscular synapses within live worms. "It so happens that the same calcium channels we studied in muscles also associate with nanometre-sized membrane domains at synapses where they modulate neuronal transmissions in both normal and disease conditions," Pinaud said. Using multi-colour CALM, his team and collaborators will probe how these tiny active zones of neurons are assembled and influence the function of calcium channels during neuron activation. In vivo single-molecule imaging identifies altered dynamics of calcium channels in?dystrophin-mutant C. elegans