Thus, a new method is necessary to induce particular events in every cell and to capture the resulting cellular dynamics by the rapid immobilization of cells at defined time points. Fourth, events like cytokinesis and endocytosis do not take place at the same time in every cell 5, 11, and thus, a particular cell that is engaged in the event must be identified from a large population of cells. Third, the fluorescence and electron micrographs cannot be precisely aligned due to tissue shrinkage caused by dehydration during the sample preparation for electron microscopy 9, 10. Second, the subcellular architecture is observed post facto 8 thus, the dynamic morphological changes cannot be captured using this approach. First, the temporal resolution is limited by how quickly the cells can be immobilized, which typically takes s - min due to the slow diffusion and reaction of fixatives 7. Although CLEM captures certain aspects of intracellular dynamics, there are four factors that limit the utility of this approach. In CLEM, cells engaged in various processes, such as cytokinesis and endocytosis 3, 4, 5, 6, are live-imaged and then processed for electron microscopy. Correlative Light and Electron Microscopy (CLEM) visualizes intracellular dynamics using light microscopy and underlying subcellular structures with electron microscopy. To overcome the limitations of light and electron microscopy, correlative microscopy techniques have been developed. Thus, it is typically not sufficient to completely understand cellular dynamics using only one imaging modality. On the other hand, while electron microscopy can delineate subcellular architecture in exquisite detail, it cannot capture cellular dynamics, because specimens must be fixed prior to imaging. However, the subcellular context is largely missing in such images because subcellular structures cannot be completely "painted" by dyes or fluorescent probes and resolved spatially and spectrally 1, 2. Dynamic trafficking events can be captured using light or fluorescence microscopy. Visualizing membrane and protein dynamics within a cell is a key step towards understanding the cell biology of particular processes. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.
To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time.
Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. We have developed a time-resolved electron microscopy technique, "flash-and-freeze," that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively.