STED microscopy: Maximum information from as many photons as possible

What are the details of the function of receptors that sit in the membrane as switching points for signal transport into the cell? Why do the distribution and activity of the mitochondria, the cell organelles responsible for energy supply, change with age? By observing the processes in the cell with molecular precision, biomedical research wants to get to the bottom of such questions and requires imaging methods with the highest spatial and temporal resolution. "We want to capture as many photons as possible and obtain the maximum information from each of them," says ACP principal scientist Prof. Dr. Michael Börsch. The physical chemist heads the Microscopy Methodology work group at the University Hospital Jena (UKJ), which conducts interdisciplinary projects on the expansion of high-resolution fluorescence imaging techniques.

For this purpose, the team and its partner groups in medicine, physics, and chemistry now have a modular STED microscope at their disposal, which was funded by the German Research Foundation (DFG) and the state of Thuringia with 900,000 euros each. STED is a variant of fluorescence microscopy, which can greatly reduce the fluorescent and, therefore, imaged area, through targeted deactivation and, thus, improve its resolution. For this deactivation and the previously necessary excitation of the dyes, the group works with eleven different lasers in the STED microscope. With its measurement setups, it, not only records the intensity of the fluorescent light, but also its service life, which suggests the chemical environment and possible reactants of the luminous molecule. "We also want to measure spectral properties and possible preferred directions of the emitted light," says Michael Börsch - "and all at the same time, and with the detection sensitivity for a single molecule". For measurements over a longer period of time in living cells, input and excitation light must protect the dyes. Michael Börsch: "This is a compromise between dissolution and detection." His compromise proposal comes with minimal deactivation intensities and is patented.

Representing the activity of ATP synthase in the mitochondria

The group intends to use this method to represent the activity of ATP synthase in the mitochondria of human cells. The enzyme repeatedly charges the energy carrier ATP, which is central to all metabolic processes, like a charger recharges a battery. This only becomes visible at a resolution below 100 nanometers. Another project is the quantitative analysis of the switching behavior of a G-protein-coupled receptor, which is involved in the regulation of many digestive processes. The STED microscope image is intended to show the cellular arrangement and the changes in the molecular structure of the activated receptor. For these projects, the group collaborates with partners in dye chemistry, as well as in applied optics and biophysics, among others. Prof. Börsch:"Our method development thrives on interdisciplinary cooperation. We are working on expanding high-resolution microscopy techniques or enabling new measurement methods for live cell analysis."