Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (SignificanceWe have developed a correlative imaging pipeline to (i) correlate 3D-FM to volume-EM data with high precision, directly bridging the FM and EM resolutions (ii) achieve high-throughput volume-CLEM by targeted EM imaging of a single organelle sized region-of-interest, pre-identified by FM (iii) link live-cell fluorescence imaging of cultured mammalian cells to high-throughput volume-CLEM (iv) quantitatively study structure-function relations at subcellular scale (v) link rare (e.g. membrane contact sites) and transient (e.g. organelle interactions) cellular events to 3D ultrastructure.The targeted volume-CLEM pipeline provides a unique prospect for multi-modal correlative intracellular analysis combining dynamic interaction (live-cell imaging), functional state (live-cell imaging), molecular localization (FM), and 3D-ultrastructure (FIB.SEM) at nanometer scale.