Microscopy enhancement shows orientation of individual DNA strands

Stanford University (CA) 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—which builds on super-resolution microscopy—could help scientists gain important insights into what goes wrong when DNA becomes damaged or when other cellular processes affect gene expression.

The research team, led by W. E. Moerner (one of the 2014 Nobel Prize in Chemistry winners for his work in super-resolution microscopy), demonstrated the technique by obtaining super-resolution images and orientation measurements for thousands of single fluorescent dye molecules attached to DNA strands (which are very long, but measure just a few nanometers across). Super-resolution microscopy, together with fluorescent dyes that attach to DNA, can be used to better visualize these strands. 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," Backer explains. "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."
 
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.
 
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.

Left: Dye molecules undergo ~400x fluorescence enhancement upon intercalation (sliding between DNA base pairs). Right: Dye concentration is reduced until single molecules are discernable. (Image courtesy of the Moerner Group)

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 their paper describing the work, the researchers demonstrated a spatial resolution of around 25 nm and single-molecule orientation measurements with an accuracy of around 5°. They also measured the rotational dynamics of single molecules with an accuracy of about 20°. 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," Backer says. "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."
 
Full details of the work appear in the journal Optica; for more information, please visit http://dx.doi.org/10.1364/optica.3.000659.

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