Dr. Jixin Chen
By Amanda Biederman
NQPI editorial intern
When a panorama camera is used to capture a landscape, the object must remain perfectly still. If the object moves during recording, a blurred, distorted image will result. This phenomenon, known as the “blurring effect,” can have a similar, problematic impact in advanced molecular visualization measurements.
Dr. Jixin Chen, Assistant Professor of Chemistry & Biochemistry and Nanoscale & Quantum Phenomena Institute member, has developed a statistical computer program to help researchers account for the blurring effect in single-molecule Förster resonance energy transfer (smFRET) measurements. The program was created in collaboration with Rice University Professors Anatoly Kolomeisky and Christy Landes, and Chen’s graduate students Joseph Pyle and Kurt Waldo Sy Piecco.
Single-molecule FRET is a classic microscopy method that uses fluorescent dyes to measure the distance between two points within a molecule. The technology can be used to track temporal changes in molecular structure, such as those of a membrane transport protein that alters its conformation in open or closed states to allow molecules to pass through it.
Using the smFRET method, researchers can calculate the time intervals in which the protein exists in either an open or closed state. This interval is known as the dwell time. By determining the average dwell time, researchers can calculate the reaction’s rate constant, a measure of how quickly these structural changes are occurring.
However, Chen said smFRET is limited by detector exposure time, which allows for molecules to be measured within a specific frame resolution. As a result, researchers are currently unable to measure relatively rapid conformational changes compared to the camera frame rate, typically at 1000 frames per second.
“You can imagine that, if you are lucky and the molecule stays in the same state within a single frame, you get one data point that is accurate,” Chen said. “But there’s a chance that an actual transition occurs within the exposure time, and you’ll have two different signals averaged over a single frame reading…. And there’s no way to distinguish them. We just have to guess.”
Chen said these “false state” readings are extremely rare in slower biological reactions, but that the risk of a false state reading becomes greater when the rate of the reaction approaches the frame resolution limit. The result is analogous to a distorted panorama photograph, which fails to reflect the actual positions of the subject.
Chen said his team’s program, which is publicly accessible, will help researchers account for the blurring effect using a Monte Carlo simulation that represents a known comparable dataset. By overlapping the experimental and simulated data, researchers can identify and account for erroneous frames within their data.
“It could be that what you analyzed already is enough on its own…but you need to further test whether the value you get from your measurement is reliable,” Chen said. “The point of this paper is to increase the frame resolution, a little bit, so the data after the analysis is more reliable.”
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