New Imaging Method Reveals Nanoscale Details about DNA

WASHINGTON–(BUSINESS WIRE)–Researchers have developed a new enhanced DNA imaging technique that can
probe the structure of individual DNA strands at the nanoscale. Since
DNA is at the root of many disease processes, the technique could help
scientists gain important insights into what goes wrong when DNA becomes
damaged or when other cellular processes affect gene expression.
The new imaging method builds on a technique called single-molecule
microscopy by adding information about the orientation and movement of
fluorescent dyes attached to the DNA strand.
W. E. Moerner, Stanford
University, USA, is the founder of single-molecule spectroscopy, a
breakthrough method from 1989 that allowed scientists to visualize
single molecules with optical microscopy for the first time. Of the 2014
Nobel Laureates for optical microscopy beyond the diffraction limit
(Moerner, Hell & Betzig), Moerner and Betzig used single molecules to
image a dense array of molecules at different times.
In The Optical Society’s journal for high impact research, Optica,
the research team led by Moerner describes their new technique and
demonstrates it by obtaining super-resolution images and orientation
measurements for thousands of single fluorescent dye molecules attached
to DNA strands.
“You can think of these new measurements as providing little
double-headed arrows that show the orientation of the molecules attached
along the DNA strand,” said Moerner. “This orientation information
reports on the local structure of the DNA bases because they constrain
the molecule. If we didn’t have this orientation information the image
would just be a spot.”
Adding more nanoscale information
A strand of DNA is a very long, but narrow string, just a few nanometers
across. Single-molecule microscopy, together with fluorescent dyes that
attach to DNA, can be used to better visualize this tiny string. Until
now, it was difficult to understand how those dyes were oriented and
impossible to know if the fluorescent dye was attached to the DNA in a
rigid or somewhat loose way.
Adam S. Backer, first author of the paper, developed a fairly simple way
to obtain orientation and rotational dynamics from thousands of single
molecules in parallel. “Our new imaging technique examines how each
individual dye molecule labeling the DNA is aligned relative to the much
larger structure of DNA,” said Backer. “We are also measuring how wobbly
each of these molecules is, which can tell us whether this molecule is
stuck in one particular alignment or whether it flops around over the
course of our measurement sequence.”
The new technique offers more detailed information than today’s
so-called “ensemble” methods, which average the orientations for a group
of molecules, and it is much faster than confocal microscopy techniques,
which analyze one molecule at a time. The new method can even be used
for molecules that are relatively dim.
Because the technique provides nanoscale information about the DNA
itself, it could be useful for monitoring DNA conformation changes or
damage to a particular region of the DNA, which would show up as changes
in the orientation of dye molecules. It could also be used to monitor
interactions between DNA and proteins, which drive many cellular
processes.
30,000 single-molecule orientations
The researchers tested the enhanced DNA imaging technique by using it to
analyze an intercalating dye; a type of fluorescent dye that slides into
the areas between DNA bases. In a typical imaging experiment, they
acquire up to 300,000 single molecule locations and 30,000
single-molecule orientation measurements in just over 13 minutes. The
analysis showed that the individual dye molecules were oriented
perpendicular to the DNA strand’s axis and that while the molecules
tended to orient in this perpendicular direction, they also moved around
within a constrained cone.
The investigators next performed a similar analysis using a different
type of fluorescent dye that consists of two parts: one part that
attaches to the side of the DNA and a fluorescent part that is connected
via a floppy tether. The enhanced DNA imaging technique detected this
floppiness, showing that the method could be useful in helping
scientists understand, on a molecule by molecule basis, whether
different labels attach to DNA in a mobile or fixed way.
In the paper, the researchers demonstrated a spatial resolution of
around 25 nanometers and single-molecule orientation measurements with
an accuracy of around 5 degrees. They also measured the rotational
dynamics, or floppiness, of single-molecules with an accuracy of about
20 degrees.
How it works
To acquire single-molecule orientation information, the researchers used
a well-studied technique that adds an optical element called an
electro-optic modulator to the single-molecule microscope. For each
camera frame, this device changed the polarization of the laser light
used to illuminate all the fluorescent dyes.
Since fluorescent dye molecules with orientations most closely aligned
with the laser light’s polarization will appear brightest, measuring the
brightness of each molecule in each camera frame allowed the researchers
to quantify orientation and rotational dynamics on a
molecule-by-molecule basis. Molecules that switched between bright and
dark in sequential frames were rigidly constrained at a particular
orientation while those that appeared bright for sequential frames were
not rigidly holding their orientation.
“If someone has a single-molecule microscope, they can perform our
technique pretty easily by adding the electro-optic modulator,” said
Backer. “We’ve used fairly standard tools in a slightly different way
and analyzed the data in a new way to gain additional biological and
physical insight.”
Paper: A.S. Backer, M.Y. Lee, W.E. Moerner, “Enhanced
DNA imaging using super-resolution microscopy and simultaneous
single-molecule orientation measurements,” Optica, 3, 6,
659 (2016). DOI: 10.1364/OPTICA.3.000659
About Optica
Optica is an open-access, online-only journal dedicated to the
rapid dissemination of high-impact peer-reviewed research across the
entire spectrum of optics and photonics. Published monthly by The
Optical Society (OSA), Optica provides a forum for pioneering
research to be swiftly accessed by the international community, whether
that research is theoretical or experimental, fundamental or applied. Optica
maintains a distinguished editorial board of more than 30 associate
editors from around the world and is overseen by Editor-in-Chief Alex
Gaeta, Columbia University, USA. For more information, visit Optica.
About The Optical Society
Founded in 1916, The Optical Society (OSA) is the leading professional
organization for scientists, engineers, students and entrepreneurs who
fuel discoveries, shape real-life applications and accelerate
achievements in the science of light. Through world-renowned
publications, meetings and membership initiatives, OSA provides quality
research, inspired interactions and dedicated resources for its
extensive global network of optics and photonics experts. For more
information, visit osa.org/100.