Conventional wisdom holds that optical microscopy can't be used to "see" something
as small as an individual molecule. But science has once again overturned conventional
wisdom. Secretary of Energy, Nobel laureate and former director of the Lawrence
Berkeley National Laboratory (Berkeley Lab) Steven Chu led the development
of a technique that enables the use of optical microscopy to image objects or
the distance between them with resolutions as small as 0.5 nanometers - one-half
of one billionth of a meter, or an order of magnitude smaller than the previous
best.
Graph on left shows that with the active feedback system off there is a resolution drift of about 0.3 pixels or 19 nanometers, but with the feedback system on resolution is maintained at better than 0.01 pixels, or about 0.64 nanometers. Image on right shows individual Cyanine (Cy) fluorescent dye molecules – Cy3 and Cy5 - used to label 20 base pairs of double-stranded DNA.
"The ability to get sub-nanometer resolution in biologically relevant
aqueous environments has the potential to revolutionize biology, particularly
structural biology," says Secretary Chu. "One of the motivations
for this work, for example, was to measure distances between proteins that form
multi-domain, highly complex structures, such as the protein assembly that forms
the human RNA polymerase II system, which initiates DNA transcription."
Secretary Chu is the co-author of a paper now appearing in the journal Nature
that describes this research. The paper is titled "Subnanometre single-molecule
localization, registration and distance measurements." The other authors
are Alexandros Pertsinidis, a post-doctoral researcher and member of Chu's
research group at the University of California (UC) Berkeley, who is now an
assistant professor at the Sloan-Kettering Institute, and Yunxiang Zhang, a
member of Chu's research group at Stanford University.
According to a law of physics known as the "diffraction limit,"
the smallest image that an optical system can resolve is about half the wavelength
of the light used to produce that image. For conventional optics, this corresponds
to about 200 nanometers. By comparison, a DNA molecule measures about 2.5 nanometers
in width.
While non-optical imaging systems, such as electron microscopes, can resolve
objects well into the subnanometer scale, these systems operate under conditions
not ideal for the study of biological samples. Detecting individual fluorescent
labels attached to biological molecules of interest using charge-coupled devices
(CCDs) - arrays of silicon chips that convert incoming light into an electrical
charge, has yielded resolutions as fine as five nanometers. However, until now
this technology has been unable to image single molecules or distances between
a pair of molecules much less than 20 nanometers.
Chu and his co-authors were able to use the same CCD-fluorescence technology
to resolve distances with subnanometer precision and accuracy by correcting
a trick of the light. The electrical charges in a CCD array are created when
photons strike the silicon and dislodge electrons, with the strength of the
charge being proportional to the intensity of the incident photons. However,
depending upon precisely where a photon hits the surface of a silicon chip,
there can be a slight difference in how the photon is absorbed and whether it
generates a measurable charge. This non-uniformity in the response of the CCD
silicon array to incoming photons, which is probably an artifact of the chip
manufacturing process, results in a blurring of pixels that makes it difficult
to resolve two points that are within a few nanometers of one another.
"We have developed an active feedback system that allows us to place
the image of a single fluorescent molecule anywhere on the CCD array with sub-pixel
precision, which in turn enables us to work in a region smaller than the typical
three pixel length-scale of the CCD non-uniformity," says Pertsinidis,
who is the lead author on the Nature paper. "With this feedback system
plus the use of additional optical beams to stabilize the microscope system,
we can create a calibrated region on the silicon array where the error due to
non-uniformity is reduced to 0.5 nanometers. By placing the molecules we want
to measure in the center of this region we can obtain subnanometer resolution
using a conventional optical microscope that you can find in any biology lab."
Chu says that the ability to move the stage of a microscope small distances
and calculate the geometric center (centroid) of the image makes it possible
to not only measure the photo-response non-uniformity between pixels, but also
to measure the non-uniformity within each individual pixel.
"Knowing this non-uniformity then allows us to make corrections between
the apparent position and the real position of the image's centroid,"
says Chu. "Since this non-uniform response is built into the CCD array
and does not change from day to day, our active feedback system allows us to
image repeatedly at the same position of the CCD array."
Pertsinidis is continuing to work with Chu and others in the group on the further
development and application of this super-resolution technique. In addition
to the human RNA polymerase II system, he and the group are using it to determine
the structure of the Epithelial cadherin molecules that are responsible for
the cell-to-cell adhesion that holds tissue and other biological materials together.
Pertsinidis, Zhang, and another postdoc in Chu's research group, Sang
Ryul Park, are also using this technique to create 3D measurements of the molecular
organization inside brain cells.
"The idea is to determine the structure and dynamics of the vesicle fusion
process that releases the neurotransmitter molecules used by neurons to communicate
with one another," Pertsinidis says. "Right now we are getting in
situ measurements with a resolution of about 10 nanometers, but we think we
can push this resolution to within two nanometers."
In a collaboration with Joe Gray, Berkeley Lab's Associate Director for
Life Sciences and a leading cancer researcher, postdocs in Chu's research
group are also using the super-resolution technique to study the attachment
of signaling molecules on the RAS protein, which has been linked to a number
of cancers, including those of the breast, pancreas, lung and colon. This research
could help explain why cancer therapies that perform well on some patients are
ineffective on others.
In addition to its biological applications, Pertsinidis, Zhang and Chu in their
Nature paper say their super-resolution technique should also prove valuable
to characterize and design precision photometric imaging systems in atomic physics
or astronomy, and allow for new tools in optical lithography and nanometrology.
This research was supported by the National Institutes of Health, the National
Science Foundation, the National Aeronautics and Space Administration, and the
Defense Advanced Research Projects Agency.