Super-Resolution Imaging Technology Locates Specific DNA Sequences

With the use of advanced optical tools and sophisticated mathematics, researchers have found a way to target the location of specific sequences along single strands of DNA, a technique that could someday help diagnose genetic diseases.

Proof-of-concept research in the Rice University (Houston, TX, USA) lab of chemist Dr. Christy Landes identified DNA sequences as short as 50 nucleotides at room temperature, an achievement she noted is unfeasible with conventional microscopes that cannot see objects that tiny, or electron microscopes that require targets to be cryogenically frozen or in a vacuum.

The technique called super-localization microscopy has been known for a while, according to Dr. Landes, but its application in biosensing is in its early stages. Scientists have seen individual double-stranded DNA molecules under optical microscopes for a long time, but the ability to visualize single-stranded DNA is a new achievement, and breaking the diffraction limit of light adds value, she noted.

The study was published online September 27, 2013, in the American Chemical Society journal Applied Materials and Interfaces. The Rice researchers call their super-resolution technique motion blur point accumulation for imaging in nanoscale topography (mbPAINT). Using it, they resolved structures as small as 30 nm by making, fundamentally, a movie of fluorescent DNA probes flowing over a known target sequence along an immobilized single strand of DNA.

The probes are labeled with a fluorescent dye that lights up only when attached to the target DNA. In the experimental setup, most would flow by unseen, but some would bind to the target for a few milliseconds, just long enough to be captured by the camera before the moving liquid pulled them away. Processing images of these brief occurrences among the background blur allows the researchers to image objects smaller than the natural diffraction limits of light-based imaging, which do not allow for the resolution of targets smaller than the wavelength of light used to illuminate them.

Even the Dr. Landes lab’s system is subject to these physical boundaries. Individual images of fluorescing probes on targets are only a pixelated blur. However, it is a blur with a bright spot, and comprehensive analysis of multiple images allows the investigators to locate that spot along the strand.

Dr. Landes reported one objective for mbPAINT is to map minute fragments of DNA. “Eventually, we’d like to get down to a couple of nucleotides,” she said. “Some diseases are characterized by one amino acid mutation, which is three nucleotides, and there are many diseases associated with very small genetic mutations that we’d like to be able to identify. We’re thinking this method will be ideally suited for diseases associated with small, localized mutations that are not possible to detect in any other inexpensive way.”

Dr. Landes envisions mpPAINT as not only more cost-effective but also able to capture information electron microscopes cannot. “One of the reasons people invented electron microscopy is to image objects smaller than light’s diffraction limit, because biomolecules such as proteins and DNAs are smaller than that,” she said. “But electron microscopy requires cryogenic temperatures or a vacuum. You can’t easily watch things react in solution. The advent of this technology allows us to see the biological processes of nano-sized objects as they happen in water, with buffers and salts, at room temperature, at body temperature or even in a cell. It’s very exciting.”

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