To better understand how proteins move and interact within a cell membrane, an imaging method called live-cell single fluorescent-molecule imaging (SFMI) can be used to tag each protein in the membrane with a fluorescent marker. The proteins are visible under a custom-built fluorescence microscope. However, the markers under the microscope photobleach over time; because of this, researchers using SFMI have only been able to observe individual molecules for about 10 s at a time. Microscope built by the Membrane Cooperativity Unit for live-cell single fluorescent-molecule imaging (SFMI). Courtesy of OIST. Researchers at Okinawa Institute of Science and Technology (OIST) have developed a way to suppress photobleaching when using SFMI. Researchers placed the cells in an environment that mimicked the actual conditions inside a living organism. To this environment, which had low concentrations of dissolved oxygen, they added the antioxidants trolox and trolox quinone. This reducing-plus-oxidizing system, combined with low oxygen concentrations, was able to strongly suppress photobleaching, with only minor effects on the cells. This approach enabled SFMI for as long as 12,000 frames (about 7 min at video rate, as compared to the general durations on the order of 10 s) with about 22-nm single-molecule localization precision. “Our method improves the observation time of fluorescent molecules by 40-fold,” said professor Akihiro Kusumi. Using the technique, researchers can record videos of protein interactions within the cell membrane without interruption. “With our method, we can now follow the movement of each individual molecular dancer for sufficiently long periods of time to understand the cellular context,” said Kusumi. With the increased time window for observation, researchers were able to study more directly and more clearly how molecules work in the cell. They studied a group of proteins, called integrins, located within regions of the membrane, known as focal adhesions. It was previously assumed that integrins were solidly fixed, but the extended observation time revealed that integrin molecules repeatedly move and stop within the focal adhesions, even migrating between adhesions. Shedding light on the behavior of focal adhesion proteins could help researchers one day develop drugs that stop cancer cells from migrating through the body, Kusumi said. The research was published in Nature Chemical Biology (doi:10.1038/s41589-018-0032-5). At time 0:00, the numbers of fluorescent molecules are about the same in both movies. The number decreases rapidly in the video on the left, which employed a conventional observation method, whereas the number of fluorescent spots decreases very slowly in the video shown on the right, which was recorded using the newly developed method. See the spots jostle around in the cell membrane. Courtesy of OIST. This movie, showing the movements of many integrin molecules, is fast-forwarded by a factor of six from real time. The focal adhesion is shown in blue, while the green spots represent single integrin molecules. The yellow lines show the trajectory of the integrin molecule. In addition to movement, it exhibited temporary immobilization, shown in red, which the researchers termed Temporary Arrest of LateraL diffusion (TALL). Courtesy of OIST.