van Rijnsoever, C., Oorschot, V. and Klumperman, J.
Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nature Methods 5: 973-980 (2008).

The visualization of fluorescent proteins in living cells is a powerful approach to study intracellular dynamics. A limitation of fluorescence imaging, however, is that it lacks fine structural information; a fluorescent spot could represent an entire organelle, an organellar subdomain or even aggregates of proteins or membranes. These limitations can be overcome by immuno–electron microscopy (immunoEM), which uniquely combines protein detection with ultrastructural detail. Electron microscopy (EM), however, requires fixation of the cells, resulting in static images with only limited information on membrane dynamics. To bridge the gap between live-cell imaging and EM, several laboratories have developed procedures for so-called correlative light-electron microscopy (CLEM). In CLEM, fluorescently tagged proteins are first imaged by light microscopy and then visualized via EM by immunolabeling or di-aminobenzidine photoconversion. None of the current CLEM approaches use the cryo-immunogold method, which is probably the most optimal method for immunoEM8. Here we introduce a CLEM approach that integrates imaging of fluorescent proteins in live cells with the cryo-immunogold technique by modifying our previously published CLEM approach on fixed cells at several steps. Briefly, gridded coverslips are coated with Formvar and gelatin, according to an optimized protocol. Cells destined for CLEM are grown on these gridded coverslips, which facilitates tracing back their location in the electron microscope. After live-cell imaging, cells are detached from the coverslip (Step 23, the process is efficient due to the Formvar and gelatin coating of the coverslip) and prepared for electron microscopy. Using this approach, we monitored the kinetics and localization at nanometer resolution of the lysosomal membrane protein LAMP-1 in live cells.