Acquisition of a Infinity Line Confocal and 3D STED Super-resolution Microscope

Lab of Alexander Matthias Walter, Department of Neuroscience, Faculty of Health and Medical Science, University of Copenhagen.

The microscope shall be used as combined confocal/STED setup to investigate how scaffolding proteins and release factors allow efficient synaptic vesicle fusion at synaptic vesicle release sites. The sub-diffraction size of these release sites (< 70 nm) requires the highest possible resolution to investigate the molecular arrangement within these sites and how molecules ‘react’ when release sites alter their activity e.g. during learning.

Our research focuses on the identification of neuronal mechanisms to achieve stable and dynamic function by plasticity modifications of the synaptic transmission strength. In the recent years we identified Unc13 as the protein that defines the position where a synaptic vesicles release their neurotransmitters that signal to neighboring cells. These release sites are positioned with nanometer precision with respect to the release inducing Ca2+ source and this is crucial for the efficacy of signal transmission and the processing of temporal information. We now identified mutations within the Unc13 gene which facilitate neurotransmitter release by altering the release probability of synaptic vesicles. One explanation for this would be a shift of Unc13 to smaller distances with respect to the Ca2+ source. Preliminary mathematical calculations suggest that distance shifts of 20 nm would be sufficient to induce such a change. Thus, to investigate whether the position of Unc13 changes with respect to the Ca2+ source upon release potentiation, a microscope is required that delivers a XY resolution of below 30 nm. The STED microscope by Abberior Instruments we want to purchase is currently is the only microscope that can achieve such a resolution.

We also need to investigate the nanoscale topology with respect to distances perpendicular to the plasma membrane (additional Z-axis). To investigate this possibility a microscope is required that allows STED imaging in 3D without a significant loss of resolution. Again, the STED microscope we want to purchase from Abberior Instruments is the only supplier of a specific technology package for super-resolution imaging in 2D as well as in 3D. This includes the easy3D STED technology, a unique proprietary technology (see patent below). Without the highest resolution or the easy3D the goal of our research project ‘The cooperative synapse’ funded by the Novo Nordisk Young Investigator award would not be achievable.

Finally, STED is a rather complicated technology. To achieve the highest possible resolution, the excitation and STED laser have to be properly aligned. Previous devices required manual alignment which is both time consuming and must be done by an expert (e.g. a specialised engineer). This support is not available at KU. Therefore, the only way to use the system at high performance is a software- and hardware based autoalignment. Abberior Instruments now provides such a solution which is absolutely unique (see patent below).

Abberior Instruments is currently the only supplier of a specific technology package including options like DyMIN STED, RESCue STED or MINFIELD STED. Those options are currently not part of our configuration, but will be purchased as future upgrade.

— German utility model DE 20 2015 105 046 (U1);

— German laid open patent application DE 10 2014 113 716 (A1);

— European laid open patent application EP 3 507 642 (A1) (To be granted 20 January 2021);

— Chinese laid open application CN 109923459 (A);

— US laid open patent application US 2019 195 800 (A1).