Functional characterization of endo-lysosomal compartments by correlative live-cell and volume electron microscopy

Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures.