Single-molecule approaches give access to the full distribution of molecule behaviors and overcome the averaging intrinsic to bulk measurement methods. They allow access to complex processes where a given molecule can have heterogeneous properties over time. Recent developments in single-molecule imaging technologies have been followed by their wide application in cellular biology and are leading to the unraveling of new mechanisms related to molecular movements. They are shaping new concepts in the dynamic equilibria of complex biological macromolecular assemblies such as synapses. These advances were made possible thanks to improvements in visualization approaches combined with new strategies to label proteins with nanoprobes. In this primer, we will review the different approaches used to track single molecules in live neurons, compare them to bulk measurements, and discuss the different concepts that have emerged from their application to synaptic biology.
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