Bridging Live-Cell Endo-Lysosomal Dynamics to 3D Electron Microscopy

Correlative live-cell imaging electron microscopy (live cell-CLEM) integrates the dynamics of fluorescently labelled molecules with ultrastructural context information at nanometer resolution [1]. Most CLEM approaches to date, however, are restricted in their EM approach by the limited volume they image and lack of 3D structural information. To overcome these limitations, we apply “focused ion beam scanning electron microscopy” (FIB-SEM) as 3D-EM approach in a live cell-CLEM setup. Here we show our first results by which we correlate live cell imaging of a single organelle to 3D structural information using resin-embedded cells [2]. Our research focuses on understanding endo-lysosomal dynamics. To visualize endo-lysosomes in live cells we combine fluorescent tagged endo-lysosomal proteins (such as LAMP1-mGFP) with endocytic tracers (such as fluorescently labeled dextran). This approach enables live-cell tracking of specific endolysosomal compartments. To visualize the 3D ultrastructure underlying the fluorescent signals, the samples are chemically fixed directly after live cell imaging, stained using heavy metals and embedded in epoxy resin, after which automated serial imaging is performed using the FIB-SEM. Figure 1 presents an example of Dextran-Alexa647 and LAMP1-mGFP labeled endo-lysosomal compartments in fixed (A) and live cells (C) and in 3D-EM (B, D). The EM images provide at ultrastructural resolution the cellular context of the compartments that were previously imaged alive. Our results are the first example of live cell-CLEM of a subcellular membrane compartment using FIBSEM. We hereby demonstrate that this method can be used to correlate the dynamics of subcellular organelles to 3D ultrastructure in large volumes.