Unravelling the structure and function of organelles and macromolecular complexes within the neuronal network poses a challenge due to their small sizes, heterogeneity, and dynamic behaviour. Conventional microscopy techniques fall short in providing the necessary resolution and speed to capture these dynamics in living cells. In the lab we develop novel optical schemes to enable gentler and faster super resolution imaging of nanoscale structures within living cells1. Leveraging these advancements, we uncovered the structural and dynamic reorganization of various organelles in neurons2,3. Moreover, we imaged synaptic vesicle dynamics after local calcium activity, and we followed processes such as vesicle endo- and exocytosis at rates up to 24 Hz4. Additionally, we provide new insights in the oligomerization process of the protein Arc, providing elucidation into its functional role in AMPA receptors endocytosis. These findings demonstrate the potential of our approaches in unravelling the dynamic subcellular architecture of neuronal cells, paving the way for deeper insights into neurobiology and synaptic function.

Labelling, visualization, and functional manipulation of biomolecules is at the forefront of chemical biology. However, selective and quantitative interrogation and analysis of biomolecules remains a challenge in the field. We employ approaches from chemistry and molecular design to tackle such issues with unconventional strategies for imaging and photocontrolling of biological targets. In one of our latest studies, we installed carbon-deuterium bonds in chromophores to yield dyes with increased fluorescent lifetimes, higher photostability, and enhanced brightness. With this in hand, we focus on the glucagon-like peptide 1 receptor (GLP1R), a class B GPCR that contributes to glucose homeostasis, and centrally, to satiety. We highlight GLP1R in its endogenous context with fluorescently labelled antagonists, the LUXendins, allowing super resolution imaging, 2-photon imaging, single particle tracking and intravital microscopy in vivo. We next genetically engineered an enzyme self-label onto GLP1R to interrogate its localization and behavior in its native context on the endogenous level. This allows the tracking of GLP1R in complex tissue settings treated with different type of drugs. Taken together, we aim to use chemistry as a flashlight to shine a spotlight on the invisible in biological systems.

The fusion of synaptic vesicles with the presynaptic active zone membrane displays the fundamental principle of neurotransmitter release – and thus of neuronal communication. Although first electron micrographs of vesicle fusion were captured more than 50 years ago, we are still far from understanding all molecular, biophysical, and kinetic aspects of the fusion process. In the here presented project, we have developed a workflow to characterize the fusion of vesicles with unprecedented structural and temporal resolution. In detail, we have combined optogenetic stimulation of neurons with plunge freezing for cryofixation, which allowed us to immobilize cellular processes only 2-5 ms after an action potential. To assess whether the stimulation was successful and to identify synapses that released neurotransmitters, we utilized the glutamate sensor iGluSnFR for cryo-confocal microscopy. Glutamate that is released during vesicle fusion can bind to iGluSnFR, which induces a conformational change and an increase in fluorescence intensity of the biosensor. During the combined optogenetic stimulation and cryofixation, iGluSnFR was arrested in its glutamate-bound state, which means that the high fluorescence intensity could be preserved and used as a marker for synaptic activity. On these active neurons, we performed cryo-electron tomography to capture and characterize synaptic vesicle fusion intermediates with best-possible structural preservation and resolution.

The nominal attainable resolution of fluorescence microscopy has reached the sub-nanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here we solve this problem by expanding the specimens and subjecting them to a fluorescence fluctuation analysis. The resulting technique, one-step nanoscale expansion (ONE) microscopy, enables the visualization of the shapes of individual membrane and soluble proteins. Conformational changes, as those undergone by the ~17 kDa protein calmodulin upon Ca2+ binding, were readily observable. ONE was also applicable to clinical samples, to describe the morphology of protein aggregates from Parkinson’s Disease patients. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine