Expansion microscopy is a recently introduced imaging technique that achieves super‐resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20–30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000‐fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25–30 nm on conventional epifluorescence microscopes. X10 provides multi‐color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high‐quality super‐resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins. Synopsis Live‐cell immunolabeling in combination with metabolic imaging reveals that ageing synaptic vesicles are inactivated after approximately 200 rounds of release in primary hippocampal cultures. Ageing synaptic vesicles are inactivated after ˜ 200 rounds of release. Inactivation is followed by a phase of inactivity before degradation. Inactivation likely precedes accumulation of damage. Inactivation may be caused by contamination of vesicles with SNAP25. SNAP25 may be involved in triggering degradation via fusion to endosomes.

The trigger for synaptic vesicle exocytosis is Ca2+, which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca2+ levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca2+ sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca2+. The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca2+ fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.