Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation

Functional imaging is the measurement of structural changes during an ongoing physiological process over time. In many cases, functional imaging has been implemented by tracking a fluorescent signal in live imaging sessions. Electron microscopy, however, excludes live imaging which has hampered functional imaging approaches on the ultrastructural level. This barrier was broken with the introduction of superfast fixation. Superfast fixation is capable of stopping and fixing membrane traffic at sufficient speed to capture a physiological process at a distinct functional state. Applying superfast fixation at sequential time points allows tracking of membrane traffic in a step-by-step fashion.This technique has been applied to track labeled endocytic vesicles at central synapses as they pass through the synaptic vesicle cycle. At synapses, neurotransmitter is released from synaptic vesicles (SVs) via fast activity-dependent exocytosis. Exocytosis is coupled to fast endocytosis that retrieves SVs components from the plasma membrane shortly after release. Fluorescent FM dyes that bind to the outer leaflet of the plasma membrane enter the endocytic vesicle during membrane retrieval and remain trapped in endocytic vesicles have been widely used to study SV exo-endocytic cycling in live imaging sessions. FM dyes can also be photoconverted into an electron-dense diaminobenzidine polymer which allows the investigation of SV cycling in the electron microscope. The combination of FM labeling with superfast fixation made it possible to track the fine structure of endocytic vesicles at 1 s intervals. Because this combination is not specialized to SV cycling, many other cellular processes can be studied. Furthermore, the technique is easy to set up and cost effective.This chapter describes activity-dependent FM dye labeling of SVs in cultured hippocampal neurons, superfast microwave-assisted fixation, photoconversion of the fluorescent endocytic vesicles, and the analysis of individual synapses after serial section 3D reconstruction of individual synapses from electron micrographs.