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Combined microscopy method may allow in vivo imaging of muscle contraction

Nov 2006
Single myosin and actin molecules can be viewed in muscle tissue

Raquel Harper

Understanding the dynamics of the interaction between myosin and actin during muscle contraction is important for both skeletal and heart muscle research, such as in the prevention and treatment of neuromuscular disorders and heart diseases. Scientists have a fairly good idea of how this interaction works from studies done in solution, but studying the interaction in real muscle tissue has proved challenging.

Now researchers at the University of North Texas Health Science Center in Fort Worth and at Chalmers University of Technology in Göteborg, Sweden, have combined total internal reflection microscopy with surface-plasmon-coupled emission to view the interaction of myosin and actin in muscle tissue.

Researchers have combined total internal reflection microscopy with surface-plasmon-coupled emission to obtain a small enough volume of molecules from a live sample of muscle tissue to view the interaction of myosin and actin. The figure shows a surface plasmon coupled emission image of a fluorescently labeled myofibril.

In solution, the muscle proteins are loosely packed, but in muscle tissue, they are extremely crowded. And this crowding has a significant effect on protein solubility and conformation — meaning that their structure and function may be quite different in muscle than in solution.

The crowding also is the reason why scientists have had a hard time studying the interaction directly in muscle tissue. The concentration of actin, especially, is much higher, making it difficult to look at a single actin molecule interacting with a single myosin molecule. To see these single molecules, scientists would have to collect data from a volume small enough to contain only a few molecules. And this has not been possible with the microscope technology currently available.

Julian Borejdo of the University of North Texas explained that not even confocal microscopes — which are a big improvement over wide-field types — can provide small enough volumes. They can limit the amount to a femtoliter and eliminate background noise, but the samples still contain thousands of molecules.

As reported in the October issue of Biophysical Journal, Borejdo and his colleagues found that they could obtain a small enough volume in a live sample that contained only a few actin molecules by fluorescently labeling the muscle and then shining a laser on it at a surface plasmon resonance angle, thereby illuminating only a small volume of the sample by evanescent wave. To define a small volume, they first homogenized a sample of muscle to obtain myofibrils and then placed the 10.0 x 1.0 x 0.50-μm size sample on a glass coverslip covered with a nanometer-thin layer of gold or silver.

They placed the coverslip on a microscope that they adapted for the procedure based on an inverted microscope from Olympus. They shined light from a 50-mW, 532-nm laser made by Coherent Inc. of Santa Clara, Calif. on the slide at the surface plasmon resonance angle, which induced surface plasmons in the metal film. The plasmons induced an evanescent wave, similar to that produced by a total internal reflection microscope but much thinner (50 nm vs. 200 nm) and more powerful.

Once the 50-nm-thick sample was illuminated, the researchers further limited the detection volume by inserting a confocal aperture into the conjugate image plane of the objective. This provided them with 2 al of sample, which contained the very few molecules of actin and myosin that they were after.

The left panel shows a magnified image of a sarcomere (composed of overlapping myosin and actin filaments) from the figure on the previous page. The black dot is a projection of the confocal pinhole on the image plane. In the schematic diagram, the fluorescently labeled actin monomers are marked red. The observational volume is about 50 nm thick, and the resulting detection volume is about 2 al.

They used a 1.65-NA objective to view the samples in water with surface plasmon coupled emission light, which required them to use coverslips made from high-refractive-index glass. They chose to use sapphire coverslips, which had a refractive index of 1.78, and this together with the thin layer of metal, enabled them to obtain images with 488- or 532-nm excitation.

Borejdo explained that another factor contributed to the small volume that they were able to obtain: There was no fluorescence emanating from the volume at the immediate interface between the metal and the sample. This, he said, is one of the reasons that surface plasmon coupled emission microscopy is different from total internal reflection microscopy. There was no fluorescence excitation at the surface between the sample and the metal coating because the fluorescence was quenched by the metal layer, meaning that the fluorescence didn’t begin until about 10 nm after the surface of the metal. At that point, it was very bright because the light was enhanced by the metal.

The researchers viewed about two layers of thin actin filaments, which corresponded to about 12 actin monomers labeled with fluorescent phalloidin. Borejdo believes that these results indicate that their long-term objective — to observe a single molecule of a contractile protein during contraction of muscle —is feasible.

Further, because the surface plasmon coupled emission method is dependent on the angle of orientation of the muscle molecule’s transition moment, the combined technique also is useful for measuring rotational motion. It is this motion that the researchers eventually want to view, so that they can watch the whole mechanochemical cycle of actin and myosin during muscle contraction.

Borejdo thinks that the combined microscopy method will be useful not only for studying muscle, but also in other systems, such as the binding of hormones to receptors on membranes.

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