Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy

Job Fermie, Nalan Liv, Corlinda ten Brink, Elly G. van Donselaar, Wally H. Müller, Nicole L. Schieber, Yannick Schwab, Hans C. Gerritsen, Judith Klumperman: Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy. In: Traffic, 19 (5), pp. 354-369, 2018.

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.

BibTeX (Download)

@article{fermie_CLEM,
title = {Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy},
author = { Job Fermie and Nalan Liv and Corlinda ten Brink and Elly G. van Donselaar and Wally H. Müller and Nicole L. Schieber and Yannick Schwab and Hans C. Gerritsen and Judith Klumperman},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/tra.12557},
doi = {10.1111/tra.12557},
year  = {2018},
date = {2018-02-16},
journal = {Traffic},
volume = {19},
number = {5},
pages = {354-369},
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.},
keywords = {correlative light-electron microscopy, endolysosomal system, focused ion beam scanning electron microscopy, organelle dynamics, time-lapse microscopy, volume electron microscopy},
pubstate = {published},
tppubtype = {article}
}