LMU researchers have found a way to assess the reliability of labeling target proteins with super-resolution fluorescence microscopy. Modern microscopy allows for detailed examination of cell processes, enabling observation of individual protein arrangements and interactions. Professor Ralf Jungmann, Chair of Molecular Physics of Life at LMU and Max Planck Fellow, explains the advancements in microscopy techniques.The team at the MPI of Biochemistry, led by biophysicist Dr. Jungmann, has recently developed the innovative RESI (Resolution Enhancement by Sequential Imaging) method. This method has the capability to enhance the resolution of fluorescence microscopy to the Ångström scale, which is significantly lower than the traditional diffraction limit of light. Crucial to this technique are DNA-conjugated marker molecules, which are precisely attached to the molecules of interest by the researchers.
In a recent publication in the journal Nature Methods, Jungmann’s team introduced a new technique for quantifying the binding effectiveness of biomarker molecules to target proteins. ”This is absolutely revolutionary,” said Jungmann.is absolutely essential for making reliable quantitative statements,” explained the physicist. Knowing the labeling efficiency allows for spatially resolved proteomics to be carried out. This method not only reveals the functions of individual proteins within a cell, but also provides information on their presence and how their quantity and behavior change under specific conditions. “However, this can only be achieved if we can accurately assess the effectiveness of the labeling process.” This is because only labeled proteins will emit light under the microscope and be visible.
Trustworthy and adaptable
Jung’s method, which has been developed, is reliable and adaptable.mann’s team has made it possible to assess target proteins by adding a reference biomarker. This biomarker glows in a different color during microscopy, allowing for successfully marked proteins to appear in two colors. They demonstrated this with the membrane protein CD86, where the reference produces a pink fluorescence and the actual marker produces a bluish one. This results in a pattern of countless pink and blue points of light. In cases where the marking did not work, only the reference lights up individually. The efficiency of the marking is calculated by comparing the ratio of double and single illuminated molecules. This method has several advantages.Advantages over previous methods for measuring binding efficiency include its ability to work both in vitro and in vivo, within intact cells. This technique is also versatile, as it can be used with various target molecules, biomarkers, and samples, and is compatible with a range of super-resolution methods. Having a reliable and widely applicable way to assess marker efficiency is essential for ensuring accurate data analysis and facilitating valid comparisons between different binders, labeling conditions, and research facilities. The authors of the study are confident in the effectiveness of the new quantification method.The expansion of their super-resolution microscope method has opened up possibilities for specific biomedical applications. This includes important quantitative detection of proteins and processes, particularly in cancer research. Understanding the interactions between proteins on the cell surface and drugs with molecular resolution is crucial for the development of new medications. The findings were published in the Journal Reference Joschka Hellmeier, Sebastian Strauss, Shuhan Xu, Luciano A. Masullo, Eduard M. Unte.rauer, Rafal Kowalewski, Ralf Jungmann. Measuring the exact labeling efficiency of individual proteins. Nature Methods, 2024; DOI: 10.1038/s41592-024-02242-5