Home Cellular science Optical imaging of dynamic interactions between molecules in a cell — ScienceDaily

Optical imaging of dynamic interactions between molecules in a cell — ScienceDaily

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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.

Exceed dynamic resolution limits

Developed by the research group of Prof. Markus Sauer (Rudolf Virchow Center and Biocenter) and Dr. Gerti Beliu (Rudolf Virchow Center) of the University of Würzburg, the new photoswitching fingerprint analysis enables optical imaging of interactions dynamics with other molecules in the cell. “So far, there is no method that reliably enables structural optical resolution in cells in the sub-10 nm range. By elucidating this underlying cause of the barrier, we have succeeded for the first time, in combination with new methods of direct labeling, to enable a cellular resolution of a few nanometers. This progress makes it possible to reveal the molecular functions and the architecture of important components of our cells”, reports Sauer.

Single molecule localization microscopy methods such as dSTORM, developed in the group of Prof. 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 immunolabeling 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 causes of the sub-10 nm resolution barrier were previously unknown. “In our publication, we have now shown for the first time that the photoswitching (blinking) rates of dyes between an on and off state are strongly affected at distances less than 10 nm due to various energy transfer processes between dyes. This results in a cluster of on-states during the first few seconds of an experiment associated with rapid photobleaching of the dyes, which makes their individual localization more difficult,” Sauer explains. “The reduced localization probability of the dyes results in therefore lower structural resolution than would be expected based on individual location accuracy. This is similar to an orchestra when all instruments play their contributions simultaneously at the start of the piece; it is not possible to choose individual soundtracks. »

The fluorescence intensity trace

However, the photoswitching fingerprint and fluorescence decay time also contain information about the number of dyes present and, due to the distance dependence of energy transfer, also information about their powerless distances. optically resolve 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. “By analyzing the photoswitching fingerprints of multimeric receptors in the plasma membrane, we were thus able for the first time to estimate the distances between receptor subunits in the range of 5-7 nm in cells and determine the number of subunits. -marked units,” says Beliu.

Visualize and understand molecular communication

In the next step, the research team intends to optimize photoswitching fingerprint analysis and use it in combination with single-molecule localization microscopy using single-molecule excitation patterns. patterns and DNA-PAINT for reliable super-resolution imaging in cells with sub-10nm resolution. This should provide new insights into the molecular organization of cellular structures, organelles and multiprotein complexes, as well as the structural elucidation of protein structures using optical methods.

The newly developed method not only offers unique insights into molecular mechanisms in infection, lipid and cancer research: photoswitchable fingerprinting also has the potential to more realistically represent the dynamics and complexity of receptors of the nervous system that are important for signal transduction at synapses. of neurons. This interaction of neurons defines our daily processes of learning and memory. “It is therefore fundamentally important to understand how this molecular orchestra assembles and functions,” says Beliu, describing the significance of these research findings.

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Materials provided by University of Würzburg. Note: Content may be edited for style and length.