Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections

摘要

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(1-3) or di-aminobenzidine photoconversion(4-7). none of the current CLEM approaches use the cryo-immunogold method, which is probably the most optimal method for immunoEM(8). here we introduce a CLEM approach that integrates imaging of fluorescent proteins in live cells with the cryo-immunogold technique (Fig. 1) by modifying our previously published CLEM approach on fixed cells(9) at several steps (Box 1). Briefly, gridded coverslips are coated with Formvar and gelatin, according to an optimized protocol (steps 1-11 and supplementary table 1 online). cells destined for CLEM are grown on these gridded coverslips (steps 12-17), which facilitates tracing back their location in the electron microscope. after live-cell imaging (steps 18-22), 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 (steps 24-32). using this approach, we monitored the kinetics and localization at nanometer resolution of the lysosomal membrane protein lamp-1 in live cells (Fig. 2).