Optical imaging of dynamic interactions between molecules in cells

Super-resolution microscopy allows fluorescence images of cells, organelles and molecular complexes to be obtained 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. This prevents, for example, the study of the molecular interactions of neurons in learning and memory processes.

Overcome dynamic resolution limit

Developed by the research groups of Prof Markus Sauer (Rudolf Virchow Center and Biocenter) and Dr Gerti Beliu (Rudolf Virchow Center) at the University of Würzburg, the new photoswitching fingerprint analysis enables optical imaging of dynamic interactions with other molecules in cells. “Until now, there was no reliable method of enabling structural optical resolution in cells in the sub-10 nm range. By elucidating the underlying cause of this barrier, we have succeeded for the first time, in combination with a new direct labeling method, to enable cellular resolution of several nanometers. These advances allow the disclosure of the molecular functions and architecture of critical components of our cells,” reports Sauer.

Single molecule localization microscopy methods such as dSTORM, developed in Prof Markus Sauer’s group, allow resolution in the 10-20 nm range. In combination with the structured illumination method, localization accuracy of up to 1 nm can be achieved for dyes. Unfortunately, this high localization precision cannot be translated into a spatial resolution of several nanometers in the cell.

The problem: current labeling methods, for example immunostaining with antibodies, cause distance errors of more than 10 nm. As a result, the size of the labeling molecules prevents nanometer-scale 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 rate of photo exchange (blinking) of a dye between the on and off states is strongly affected at distances below 10 nm due to various energy transfer processes between the dyes. This resulted in a cluster of active states during the first seconds of the experiment associated with rapid bleaching of the dye, which made localization of individual dyes more difficult,” explains Sauer. “The reduced dye localization probability therefore results in poorer structural resolution than would be expected based on individual localization precision. This is similar to an orchestra when all the instruments play their contributions simultaneously at the beginning of the piece; it is impossible to select individual sound tracks.”

Traces of fluorescence intensity

However, photo-switching fingerprints and fluorescence decay times also contain information about the amount of dye present and, due to the dependence of the energy transfer distance, also information about their distance without being able to optically resolve individual dyes. By inserting unnatural amino acids into multimeric membrane receptors via extension of the genetic code followed by labeling of bioorthogonal clicks with small fluorescent dyes, the Würzburg research group can now show in a next step how site-specific labeling of proteins in cells can be achieved without distance-by-distance error. sub-10 nm. “By analyzing the photoswitching fingerprint of multimeric receptors in the plasma membrane, we were able for the first time to estimate the distance between receptor subunits in the 5-7 nm range in cells and determine the number of labeled subunits,” Beliu said.

Visualize and understand molecular communication

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

The newly developed method not only offers unique insights into molecular mechanisms in infection, lipids, and cancer research: Transferring photo fingerprints also has the potential to more realistically represent the dynamics and complexity of receptors in the nervous system that are important for signal transduction at synapses. of neurons. The interactions of these neurons define our everyday learning and memory processes. “It is therefore fundamentally important to understand how these molecular orchestras assemble and function,” said Beliu, explaining the importance of the study’s findings.

Reference:

1. Dominic A. Helmerich, Gerti Beliu, Danush Taban, Mara Meub, Marcel Streit, Alexander Kuhlemann, Sören Doose, Markus Sauer. Photoswitching fingerprint analysis passed the 10-nm resolution barrier. Natural Method, 2022; DOI: 10.1038/s41592-022-01548-6

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