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Pregnancy Timeline by SemestersFetal liver is producing blood cellsHead may position into pelvisBrain convolutions beginFull TermWhite fat begins to be madeWhite fat begins to be madeHead may position into pelvisImmune system beginningImmune system beginningPeriod of rapid brain growthBrain convolutions beginLungs begin to produce surfactantSensory brain waves begin to activateSensory brain waves begin to activateInner Ear Bones HardenBone marrow starts making blood cellsBone marrow starts making blood cellsBrown fat surrounds lymphatic systemFetal sexual organs visibleFinger and toe prints appearFinger and toe prints appearHeartbeat can be detectedHeartbeat can be detectedBasic Brain Structure in PlaceThe Appearance of SomitesFirst Detectable Brain WavesA Four Chambered HeartBeginning Cerebral HemispheresFemale Reproductive SystemEnd of Embryonic PeriodEnd of Embryonic PeriodFirst Thin Layer of Skin AppearsThird TrimesterSecond TrimesterFirst TrimesterFertilizationDevelopmental Timeline
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Home | Pregnancy Timeline | News Alerts |News Archive Oct 15, 2013

 


Diagram of  laser beam capturing a movie of fluorescent DNA.










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Using motion blur to sharpen DNA mapping

With high-tech optical tools and sophisticated mathematics, Rice University researchers have found a way to pinpoint the location of specific sequences along single strands of DNA, a technique that could someday help diagnose genetic diseases through location of exact gene sequences.

Proof-of-concept experiments in the Rice lab of chemist Christy Landes identified DNA sequences as short as 50 nucleotides at room temperature, a feat she said is impossible with standard microscopes that cannot see targets that small, or electron microscopes that require targets to be in a vacuum or cryogenically frozen.

The technique called “super-localization microscopy” has been known for a while, Landes said, but its application in biosensing is just beginning. Scientists have seen individual double-stranded DNA molecules under optical microscopes for years, but the ability to see single-stranded DNA is a new achievement, and breaking the diffraction limit of light adds value, she said.

The work by Landes, Rice postdoctoral associate Jixin Chen and undergraduate student Alberto Bremauntz is detailed 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). With it, they resolved structures as small as 30 nanometers (billionths of a meter) by making, essentially, 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 targeted DNA.

In the experimental setup, most probes flow by unseen, but some bind to their 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 events amidst the background blur allows researchers to image objects smaller than the limits of light-based imaging, which does not allow for resolution of targets smaller than the wavelength of light used to illuminate them.


Even the Landes lab’s system is subject to these physical limitations. Individual images of fluorescing probes on targets are just a pixelated blur. But it’s a blur with a bright spot, and careful analysis of multiple images allows the researchers to pinpoint that spot along the strand.

“The probes are moving so fast that in real time, all we would see with the camera is a line,” Chen said. But when the camera firing at 30-millisecond intervals happened to catch a bound probe, it clearly stood out. The probes sometime picked out two sequences along a strand that would have been seen as a single blur via regular fluorescent microscopy.

Landes said one goal for mbPAINT is to map ever-smaller fragments of DNA. “Eventually, we’d like to get down to a couple of nucleotides,” she adds. “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,” she said.

Landes sees 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. 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,”

Christy Landes, chemist, Rice University


Abstract
We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as “motion blur” point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.

Rice graduate students Lydia Kisley and Bo Shaung are co-authors of the paper.

The National Science Foundation, the Welch Foundation and the National Institutes of Health supported the research.

Original press releas: http://news.rice.edu/2013/10/04/scientists-use-blur-to-sharpen-dna-mapping-2/