Blurred images bring clarity
Technique reveals rotational as well as translational dynamics of molecules
Consider an actor
on a stage. If he takes a few steps to his left, it is fairly easy to determine
how far he moves and in what direction — the tools with which to do so are
readily available. But then, as he continues, he begins to twist his body and spin
his arms. It’s a mélange of movement, as revealing of his motivations
as the steps he takes. Still, how do you measure that?
Researchers at the University of Illinois at
Urbana-Champaign, at Research Institute Jülich in Germany, at Case Western
Reserve University in Cleveland, and at the University of Pennsylvania in Philadelphia
were faced with a similar problem when looking at the movement of biological macromolecules,
such as myosin V, that exhibit both translational and rotational movement. A variety
of techniques — for example, optical trapping and fluorescence imaging with
1-nm accuracy — are available for measuring the step sizes of molecular motors.
“But these methods don’t
tell much about the rotational dynamics of molecular motors … ” said
Erdal Toprak, a graduate student at the University of Illinois. “A method
that can measure rotational dynamics effectively would be very useful to our lab.”
Toprak and colleagues in Paul R. Selvin’s
group at Urbana-Champaign therefore decided to try the defocused orientation and
position imaging technique, which had been successfully demonstrated in 1999 by
Andrew P. Bartko and Robert M. Dickson and theoretically solved in 2003 by Martin
Böhmer and Jörg Enderlein of Research Institute Jülich. This technique
provides information about the angular distribution of a single molecule’s
fluorescence emission by defocusing the image acquired with a wide-field epifluorescence
or a total internal reflection microscope. The intensity distribution of the resulting
blurred image contains the relevant information.
The intensity distribution of blurred images obtained by defocusing a microscope contains information about the angular distribution of the molecules under observation. Shown here are images
of the 3-D orientations of quantum dots acquired with the defocusing technique.
Images courtesy of Erdal Toprak.
The method allows imaging of rotational
dynamics, which fluorescence imaging at 1 nm, for instance, does not. At the same
time, though, it does not always identify the molecule’s position as accurately
because the image is spread over a greater number of pixels. For this reason, the
investigators came up with a way to switch back and forth between the focused and
defocused imaging, providing information about position and orientation with considerable
In the April 25 issue of PNAS,
they reported that they used this combined approach to track the translational and
rotational dynamics of fluorescently labeled myosin V as the proteins moved along
actin — long, rodlike proteins involved in muscular contraction. Myosin V
is a motor protein that hauls cargo along actin in a stepwise, “hand-over-hand”
manner, following a helical path around the protein as it does so. It was the perfect
choice for demonstrating the technique, Toprak noted, because its “lever arm”
— which drags it forward as it moves, alternately leading and trailing —
is known to tilt with respect to actin.
The researchers observed 32 molecules
during the study, using different patterns of defocused and focused imaging (and
65 molecules using defocused orientation and position imaging only). In one experiment,
for example, they measured displacement and 3-D orientation of the molecules with
repeated cycles of five consecutive defocused images and three consecutive focused
images. They used an exposure time of 0.66 s per frame. In all cases, the sample
was moved about 500 nm from the optimal focus position.
The defocusing technique provides information about the translational and rotational
dynamics of molecules based on defocused images, shedding light on the dynamics
of a myosin V molecule as it moves across actin.
In all of the experiments, the scientists
used a total internal reflection microscope setup based on an inverted microscope
made by Olympus America Inc. of Melville, N.Y., with a 1.6x magnification unit
and an infinity-corrected 100x, 1.45-NA oil-immersion objective. A 532-nm diode-pumped
Nd:YAG laser made by CrystaLaser LC of Reno, Nev., provided excitation, and a back-thinned
CCD camera from Andor Technology of South Windsor, Conn., captured the images. They
controlled the distance between the sample and objective (and thus defocusing) with
a piezoelectric Z-axis sample stage made by Mad City Labs Inc. of Madison, Wis.
Image acquisition and defocusing were synchronized using custom-written software.
The researchers unveiled several features
of the lever arm dynamics and translocation of the myosin, showing, for example,
that all of the moving myosin exhibited lever arm tilts and that the tilting and
stepping events occurred simultaneously. “We can distinguish the trailing
and leading lever arm states of myosin,” Toprak said. “I believe that
this may help to understand how the two lever arms communicate with each other.”
The technique could be used to study
any number of biological molecules that rotate, and possibly even membrane and motor
proteins. It might also be applied to visualize the alignments of fluorescent nanocrystals.
Other methods, such as single-molecule
fluorescence polarization microscopy, can measure the 3-D orientations of fluorophores
with a time resolution of about 40 ms. With normal excitation powers, defocused
orientation and position imaging offers a resolution of 100 ms for quantum dots
and 0.5 s for conventional fluorescent dyes.
However, these other methods generally
have angular degeneracy issues. For example, single-molecule fluorescence polarization
microscopy detects the 3-D orientation of dipoles based on their absorptions and
emissions. However, dipoles with orientations 180° apart can yield
exactly the same absorption and emission and thus can be impossible to distinguish.
Also, the setups are often costly and difficult to build. “Our method is relatively
easy and [inexpensive] to use, and we have no angular degeneracy,” Toprak
The researchers continue to develop
the technique, working to increase its time resolution and positional accuracy.
They also are planning to apply it to other motor proteins.
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