Single organelle dynamics linked to 3D structure by correlative live‐cell imaging and 3D electron microscopy |
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Authors: | Job Fermie Nalan Liv Corlinda ten Brink Elly G. van Donselaar Wally H. Müller Nicole L. Schieber Yannick Schwab Hans C. Gerritsen Judith Klumperman |
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Affiliation: | 1. Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands;2. Section Molecular Biophysics, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands;3. Section Cryo‐EM, Department of Chemistry, Utrecht University, Utrecht, The Netherlands;4. Electron Microscopy Core Facility, EMBL Heidelberg, Heidelberg, Germany |
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Abstract: | Live‐cell correlative light‐electron microscopy (live‐cell‐CLEM) 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 3‐dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB‐SEM) in a modular live‐cell‐CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal‐associated membrane protein 1‐green fluorescent protein (LAMP‐1‐GFP), analyzed the dynamics of individual GFP‐positive spots, and correlated these to their corresponding fine‐architecture and immediate cellular environment. By FIB‐SEM 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 FIB‐SEM, which significantly reduces time required for image acquisition and data processing. ![image](//wol-prod-cdn.literatumonline.com/cms/attachment/e9fae2ca-f042-42e3-afa2-7408932f87c2/tra12557-gra-0001.png) |
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Keywords: | correlative light‐electron microscopy endolysosomal system focused ion beam scanning electron microscopy organelle dynamics time‐lapse microscopy volume electron microscopy |
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