The researchers characterize their new technique as a neat solution to the
"needle in a haystack" problem of nanoscale microscopy, but it's more
like the difference between finding the coffee table in a darkened room either
by walking around until you fall over it, or using a flashlight.
In a new paper,* a group from JILA—a joint venture of the National
Institute of Standards and Technology (NIST) and the University of Colorado—finds
tiny assemblies of biomolecules for subsequent detailed imaging by combining
precision laser optics with atomic force microscopy.
The atomic force microscope (AFM) has become one of the standard tools of nanotechnology.
The concept is deceptively simple. A needle—not unlike an old-fashioned
phonograph stylus, but much smaller with a tip at most only a couple of atoms
wide—moves across the surface of the specimen. A laser measures tiny deflections
of the tip as it is pushed or pulled by atomic scale forces, such as electrostatic
forces or chemical attraction. Scanning the tip back and forth across the sample
yields a three-dimensional image of the surface. The resolution can be astonishing—in
some cases showing individual atoms, a resolution a thousand times smaller than
the best optical microscopes can achieve.
Such amazing sensitivity incurs a technical problem: if your probe can image
an object of, say, 100 square nanometers, how exactly do you find that object
if it could be nearly anywhere on a microscope stage a million times that size?
That's not an unusual case in biological applications. The brute-force answer
is, you scan the probe back and forth, probably at a higher speed, until it
runs into something interesting. Like the coffee table in the dark, this has
problems. The AFM tip is not only very delicate and easy to damage, but it can
be degraded by picking up unwanted atoms or molecules from the surface. Also,
in the biosciences, where the AFM is becoming increasingly important, research
specimens usually are "soft" things like proteins or membranes that
can be damaged by an uncontrolled collision with the tip. One solution has been
to "label" the target molecule with a small fluorescent compound or
quantum dot, so that it lights up and is easy to find, but that means chemically
altering the subject, which may not be desirable.
Instead, the JILA team opted to use a flashlight. Building upon an earlier
innovation for stabilizing the position of an AFM tip, the group uses a tightly
focused, low-power laser beam to optically scan the area, identifying target
locations by minute changes in the scattered light. This laser is scanned across
the sample to form an image, analogous to forming an AFM image.
The same laser—and detection technique—is used to locate the AFM
tip. Hence, the laser serves as a common frame of reference and it's relatively
straightforward to align the optical and the AFM image. In experiments with
patches of cell membrane from single-cell organisms,** the group has demonstrated
that they can locate these protein complexes and align the AFM tip with a precision
of about 40 nanometers. Relying solely on scattered light, their technique requires
no prior chemical labeling or modification of the target molecules.
"You solve a couple of problems," says NIST physicist Thomas Perkins.
"You solve the problem of finding the object you want to study, which is
sort of a needle in a haystack problem. You solve the problem of not contaminating
your tip. And, you solve the problem of not crashing your tip into what you
were looking for. This prevents damaging your tip and, for soft biological targets,
not damaging your sample." And, he says, it's much more efficient. "From
a practical perspective, instead of my grad student starting to do real science
at 4 p.m., she can start doing science at 10 a.m."
* A.B. Churnside, G.M. King and T.T. Perkins. Label-free optical imaging of
membrane patches for atomic force microscopy. Optics Express. Vol. 18, No. 23.
Nov. 8, 2010.
** The team used "purple membrane," which is cell membrane from certain
single-cell organisms and contains bacteriorhodopsin, a protein that captures
light energy. Bacteriorhodopsin is embedded in purple membrane and is a common
protein for research in the biosciences.