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Neurons are polarized cells that encode information in the nervous system viaelectrochemical connections named synapses. The tuning of synapticconnections is enabled by a plastic protein trafficking system which operates atthe nanoscale to finely tweak the neuronal ultrastructure. Our understanding ofthe neuronal biology has certainly benefited from the advent of live-cellcompatible fluorescence techniques able to reach the molecular level. However,the neuronal trafficking system involves molecular complexes, from organelles tosynaptic modulators, which act with varying dynamics at differentspatiotemporal scales. A single technique struggles to portrait these complexphenomena since it is hard to combine molecular resolution, speed, and lowphototoxicity. Hence, their investigation often demands, together with technicaladvancements, the combination of advanced fluorescence methods withcomplementary features. In this thesis, I explore the neuronal protein traffickingsystem at the molecular scale applying cutting-edge fluorescence microscopy andspectroscopy techniques.The relationship between the geometry and dynamics of the tubular endoplasmicnetwork and the sub-compartment size of neurons is investigated using acombination of STED and parallelized RESOLFT microscopy. In addition, thethree-dimensional dynamic interaction between tubular endoplasmic reticulumand mitochondria is described.The basal activity-driven recycling of synaptic vesicles is, for the first time,monitored via event-triggered STED, an automated method able to initiate STEDimaging upon detection of events such as calcium spikes.Insights into the post-synaptic reorganization of scaffolding and skeletal proteinsupon stimulation is gained by extending the live-cell super-resolution throughputto all the dimensions with multi-foci and 3D parallelized RESOLFT.Lastly, the molecular states of Activity-Regulated Cytoskeleton-Associatedprotein (Arc) involved in distinct aspects of neuronal protein trafficking a, Neuroner är polariserade celler som kodar information i nervsystemet viaelektrokemiska kopplingar genom synapser. Inställningen av synaptiskaanslutningar möjliggörs av ett plastproteinhandelssystem som fungerar pånanoskala för att finjustera den neuronala ultrastrukturen. Vår förståelse av denneuronala biologin gynnades verkligen av tillkomsten av levande cell-kompatiblafluorescenstekniker som kan nå den molekylära nivån. Emellertid involverar detneuronala människohandelssystemet molekylära komplex, från organeller tillsynaptiska modulatorer, som verkar med distinkt dynamik på varierandespatiotemporala skalor. En enda teknik kämpar för att porträttera dessakomplexa fenomen eftersom det är svårt att kombinera molekylär upplösning,hastighet och mildhet. Därför kräver deras undersökning ofta, tillsammans medtekniska framsteg, kombinationen av fluorescensmetoder med komplementäraegenskaper. I avhandlingen utforskar jag det neuronala proteinhandelssystemeti molekylär skala med användning av distinkta banbrytandefluorescensmikroskopi och spektroskopitekniker. Den neuronala tubuläraendoplasmatiska nätverksgeometrin och dynamiken var relaterad tillunderavdelningens storlek genom att kombinera STED och parallelliseradRESOLFT-mikroskopi. Dessutom beskrevs den tredimensionella dynamiskainteraktionen mellan tubulärt endoplasmatiskt retikulum och mitokondrier.Den basalaktivitetsdrivna återvinningen av synaptiska vesiklar övervakades förförsta gången via händelseutlöst STED, en automatiserad metod som kan initieraSTED-avbildning vid upptäckt av händelser som kalciumspikar.Insikter i den postsynaptiska omorganiseringen av byggnadsställningar ochskelettproteiner vid stimulering erhölls och utökade genomströmningen avlevande cellers superupplösning till alla dimensioner med multifoci och 3DparallelliseradRESOLFT.Slutligen studerades de molekylära tillstånden av Activity-RegulatedCytoskeleton-Associated Protein (Arc), involverat i distinkta aspekter avneuronal proteinhandel. Våra obs, QC 2023-11-17

Super-resolution fluorescence microscopy holds tremendous potential for discovery in neuroscience. Much of the molecular machinery and anatomic specializations that give rise to the unique and bewildering electrochemical activity of neurons are nanoscale by design, ranging somewhere between 1 nm and 1 lm. It is at this scale where most of the unknown and exciting action is and where cell biolo-gists flock to in their dreams, but it was off limits for light microscopy until recently. While the optical principles of super-resolution microscopy are firmly established by now, the technology continues to advance rapidly in many crucial areas, enhancing its perform-ance and reliability, and making it more accessible and user-friendly, which is sorely needed. Indeed, super-resolution microscopy tech-niques are nowadays widely used for visualizing immunolabeled protein distributions in fixed or living cells. However, a great potential of super-resolution microscopy for neuroscience lies in shining light on the nanoscale structures and biochemical activities in live-tissue settings, which should be developed and harnessed much more fully. In this review, we will present several vivid examples based on STED and RESOLFT super-resolution microscopy, illustrating the possibilities and challenges of nano-imaging in vivo to pique the interest of tech-developers and neurobiologists alike. We will cover recent technical progress that is facilitating in vivo applications, and share new biological insights into the nanoscale mechanisms of cellular communication between neurons and glia., QC 20221209

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastictargeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers 45 - 65 nm resolution of large fields of view in a few seconds. In MoNaLISA, engineered light patterns strategically confine the fluorescence to sub-diffracted volumes in a large area and provide optical sectioning, thus enabling volumetric imaging at high speeds. The optical setup presented in this paper extends the degree of parallelisation of the MoNaLISA microscope by more than four times, reaching a field-of-view of (100 - 130 mu m)(2). We set up the periodicity and the optical scheme of the illumination patterns to be power-efficient and homogeneous. In a single recording, this new configuration enables super-resolution imaging of an extended population of the post- synaptic density protein Homer1c in living hippocampal neurons., QC 20230426

The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites. Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at similar to 50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites., QC 20210710

Arc (or Arg3.1), Activity Regulated-Cytoskeleton associated-protein is pivotal to mediate plastic responses in neuronal cells. In vitro and in vivo studies suggest its ability to form high- and low-order oligomers which are potentially involved in neuronal trafficking. Despite its important function, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we take advantage of several complementary advanced fluorescence microscopy and spectroscopy techniques to observe and quantify Arc oligomeric states in cellular environment especially in the synapses. In cells, we uncovered Arc-Arc intermolecular interactions, Arc tendency to form liquid condensates and to interact with lipid bilayers. High-order oligomers are found to localize at the excitatory synaptic compartment and to directly affects AMPA receptor surface levels. Together, our observations support the model by which Arc oligomerization mediates plasma- membrane negative inward curvature favoring AMPA receptors endocytosis., QC 20231115

The observation of protein organization during cellular signalling calls for imaging methods with increased spatial and temporal resolution. STED nanoscopy can access dynamics of nanoscale structures in living cells. However, the available number of recordable frames is often limited by photo-bleaching. Here, we present an automated method, event-triggered STED, which instantly (< 40 ms) images synaptic proteins with high spatial and temporal resolution (~30 nm, 2.5 Hz) in small regions upon and at the site of local calcium sensing., QC 20211124