@article{Fokkema2018,

title = {Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy},

author = {Jantina Fokkema and Job Fermie and Nalan Liv and Dave J. van den Heuvel and Tom O. M. Konings and Gerhard A. Blab and Andries Meijerink and Judith Klumperman and Hans C. Gerritsen},

url = {https://doi.org/10.1038/s41598-018-31836-1},

doi = {10.1038/s41598-018-31836-1},

year  = {2018},

date = {2018-09-11},

journal = {Scientific Reports},

volume = {8},

number = {1},

pages = {13625},

abstract = {In this work, gold nanoparticles coated with a fluorescently labelled (rhodamine B) silica shell are presented as fiducial markers for correlative light and electron microscopy (CLEM). The synthesis of the particles is optimized to obtain homogeneous, spherical core-shell particles of arbitrary size. Next, particles labelled with different fluorophore densities are characterized to determine under which conditions bright and (photo)stable particles can be obtained. 2 and 3D CLEM examples are presented where optimized particles are used for correlation. In the 2D example, fiducials are added to a cryosection of cells whereas in the 3D example cells are imaged after endocytosis of the fiducials. Both examples demonstrate that the particles are clearly visible in both modalities and can be used for correlation. Additionally, the recognizable core-shell structure of the fiducials proves to be very powerful in electron microscopy: it makes it possible to irrefutably identify the particles and makes it easy to accurately determine the center of the fiducials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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 = {},

pubstate = {published},

tppubtype = {article}

}