4 research outputs found
Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy
Liveâcell correlative lightâelectron microscopy (liveâcellâCLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3âdimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIBâSEM) in a modular liveâcellâCLEM pipeline for a single organelle CLEM. We transfected cells with lysosomalâassociated membrane protein 1âgreen fluorescent protein (LAMPâ1âGFP), analyzed the dynamics of individual GFPâpositive spots, and correlated these to their corresponding fineâarchitecture and immediate cellular environment. By FIBâSEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIBâSEM, which significantly reduces time required for image acquisition and data processing