12 November 2023 Looking at single molecules in living cells Back to news Researchers from the group of Marvin Tanenbaum at the Hubrecht Institute and the group of Cees Dekker at Delft University of Technology have developed a way to visualize single molecules inside living cells under the microscope. This way, individual molecules and their behavior can now be studied in living cells, something that was very difficult before. The results of the study were published in the scientific journal ACS Nano on the fourth of October. Until now it has been difficult to look at single molecules in living cells, simply because the volume of a cell is so small, and there are so many molecules, that it is difficult to see the fluorescence of one single molecule over the background fluorescence of all molecules with current microscope techniques. Being able to see and track these individual models will be valuable for understanding the molecular mechanisms that underly many biological processes. To be able to see individual molecules in living cells, the researchers made a very thin metal film with tiny holes, called zero-mode waveguide nanowells, in it. This reduces the volume in which the molecules are observed. This method had been used before for purified molecules, and was now tested with live cells. The cells form stable protrusions into the tiny holes, allowing for the observation of individual molecules over time. The researchers found that using these nanowells greatly reduced the background fluorescence, which enabled them to visualize both individual molecules that are bound to the membrane of the cells, and individual molecules that are in the cytoplasm. Sora Yang, one of the researchers in this study: “Our new technique opens up exciting possibilities for single-molecule fluorescence microscopy in live cells. By overcoming the concentration barrier, a longstanding obstacle in this field, we can now watch individual molecules in real-time within living cells, which offers fascinating opportunities to explore biological processes.” Cells are grown on zero-mode waveguide nanowells, depicted by a cell on a black surface with dots. The dots represent the fluorescent single molecules in the nanowells (upper left). Next, a zoom in of the cell surface with a protrusion into the nanowell, which allows for the visualization of a single membrane bound fluorescent molecule despite a background of many fluorescent molecules on the rest of the membrane (right). Last, graph that shows the presence of fluorescent molecules in a nanowell over time, something that could not be made using TIRF microscopy (bottom left). Credit: Sora Yang, copyright Hubrecht Institute Publication Zero-Mode Waveguide Nanowells for Single-Molecule Detection in Living Cells. Sora Yang#, Nils Klughammer#, Anders Barth#, Marvin E. Tanenbaum*, and Cees Dekker*. ACS Nano 2023. # and * these authors contributed equally to the study Marvin Tanenbaum is group leader at the Hubrecht Institute, professor of Gene Expression Dynamics at TU Delft and Oncode Investigator.
