What an inspiring morning at the CatCat Symposium! Thank you so much for inviting me @gallegolab.bsky.social and @felixmendu.bsky.social, it's been a pleasure visiting you in Barcelona 😊

Cryo-confocal microscopy of a neuron expressing the glutamate sensor iGluSnFR 🥶 The neuron was optogenetically stimulated and subsequently plunge-frozen to preserve the high fluorescence intensity indicating neurotransmitter release. #FluorescenceFriday

... but also between vesicles and cytoskeletal fibers likely resembling actin.

We did not only observe filamentous connectors at vesicles but also between vesicles and fusion events....

We further quantified distributions and tethering of synaptic vesicles at the active zone. Tether numbers closely correlated to the distance between vesicle and membrane, but not to their respective fusion readiness.

Overall, we analyzed more than 400 tomograms of synapses and identified more than 50 vesicle fusion intermediates. We classified and morphometrically characterized them.

Beyond that, we were able to correlate the cryo-fluorescent signal of iGluSnFR3 with a tomogram of a neuronal synapse. The arrowhead points to a putative vesicle fusion event.

We confirmed successful stimulation via cryo-confocal microscopy of the glutamate sensor iGluSnFR3. With this experiment, we also proved that fast fluorescent biosensors can be used under cryogenic conditions.

We developed a workflow for time-resolved in situ cryo-ET by coupling optogenetics with subsequent plunge freezing of neurons. Our setup allowed us to stimulate neurons only a few milliseconds before cryofixation.

Excited to share our new preprint "Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons" www.biorxiv.org/content/10.1...

We combine optogenetic stimulation of neurons with in situ cryo-ET to characterize membrane dynamics during synaptic vesicle fusion ❄️🔬⏱️