- Combined microscopy method may allow in vivo imaging of muscle contraction
Single myosin and actin molecules can be viewed in muscle tissue
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
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
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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