Super-resolution microscopy makes it possible to acquire fluorescence images of cells, organelles and molecular complexes with unprecedented spatial resolution. However, this resolution is not sufficient to resolve proteins as small as a few nanometers and their interactions with other molecules or the architecture of protein complexes. It prevents, for example, the study of the molecular interaction of neurons in the processes of learning and memory.
Exceeding the limits of dynamic resolution
Developed by the research group by Prof. Markus Sauer (Rudolf Virchow Heart and Biocenter) and Dr. Gerti Beliu (Rudolf Virchow Heart) from the University of Würzburg. “So far, there is no method that reliably provides structural optical resolution in cells in the sub-20nm range. By elucidating this underlying lead to the barrier, we succeeded for the first time, in combination with new immediate labeling methods, in enabling a cellular resolution of a few nanometers. This progress reveals the molecular functions and architecture of important components of our cells,” reports Sauer.
Single molecule localization microscopy methods such as dSTORM, developed in the group of Professor Markus Sauer, allow resolutions of the order of 10 to 20 nm. In combination with structured illumination methods, localization accuracies of up to 1 nm could be achieved for dyes. Unfortunately, this high localization precision could not be translated into a spatial resolution of a few nanometers in the cells.
The problem: current labeling methods, for example l immunostaining with an antibody, cause a spacing error of more than 10 nm. As a result, the size of the labeling molecules impedes nanoscale resolution. Other results in the sub-10nm resolution barrier were previously unknown. “In our publication., which makes their individual location more difficult,” says Sauer. “The reduced localization probability of the dyes therefore results in lower structural resolution than would be expected based on individual localization accuracy. This is similar to an orchestra where all devices play their contributions simultaneously at the beginning of the piece it is difficult to choose the individual soundtracks. »
The fluorescence intensity trace
However. due to the length dependence of the energy transfer, also information about their distances without being able to optically resolve the individual dyes. By incorporating unnatural amino acids into multimeric membrane receptors by expanding the genetic code followed by bioorthogonal click-tagging with small fluorescent dyes, the Würzburg research groups have now been able to show in the next step how a Site-specific labeling of proteins in cells can be achieved without spacing errors with distances less than 10 nm. we were thus able for the first time to estimate the distances between the receptor subunits in the 5-7 nm range in the cells and determine the number of labeled subunits”, explains Beliu.