In a new imaging technique developed at Duke University in collaboration with Oak Ridge National Laboratory, short strands of DNA were labeled with fluorescent, temporal “barcodes” for observing biochemical activity at the molecular scale. This time-based approach could improve optical multiplexing of biochemical events. Here is how the technique works: During the interaction between two complementary strands of DNA as they collide in a solution, one DNA strand is attached to a molecule that the researchers want to study. The other strand is free-floating. The free-floating strand carries a fluorescent dye that lights up when the two strands pair up. When the strands separate, the fluorescence disappears and the free-floating strand goes dark. When viewed under a microscope over time, the binding and unbinding of the DNA strands create a distinct flashing pattern that, when decoded, acts as a fingerprint. Since each DNA strand is programmed to exhibit different hybridization kinetics, each strand’s fluorescent time trace, referred to as the temporal barcode, will be unique. These tiny points of light might look like stars twinkling in the sky. But in reality they're different molecules of light-up DNA, blinking on and off as they bind and unbind under a microscope. Courtesy of Shalin Shah, Duke University. Traditional techniques use different color dyes to distinguish molecules, or use one dye color but different DNA sequences, and perform imaging in steps, washing the dye off one target before moving on to the next sample. Unlike previous single-color methods, the Duke team’s approach increases the number of different signals that can be identified using a single dye color, without the use of multiple DNA sequences. In the new approach, the sequence of the free-floating DNA strand remains the same, and adjustments are made to the DNA strand that is attached to the molecule under observation. This allows flashing patterns to be produced with different frequencies, durations, and brightness. Based on computer simulations, the researchers suggest that it is theoretically possible to use their method to distinguish as many as 56 different molecules simultaneously using a single dye. According to the researchers, their technique costs less than other methods, and the temporal barcodes do not fade over time under the glare of the microscope. “Our goal is to develop an economical and simple, yet powerful method,” researcher Shalin Shah said. “The temporal intensity signals emitted are distinct and can act as a fingerprint.” The research was published in ACS Synthetic Biology (http://dx.doi.org/10.1021/acssynbio.9b00010). In a paper published March 21, 2019, in Nano Letters (https://pubs.acs.org/doi/10.1021/acs.nanolett.9b00590), the team reported on the lab results of tests to their approach.