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

Fermie, Job; Liv, Nalan; Brink, Corlinda ten; Donselaar, Elly G. van; Müller, Wally H.; Schieber, Nicole L.; Schwab, Yannick; Gerritsen, Hans C.; Klumperman, Judith

doi:10.1111/tra.12557

Cited by 85 publications

(95 citation statements)

References 92 publications

(169 reference statements)

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“…Finally, we explored the endolysosomal pathway, the compartments of which are notoriously sensitive to artifacts of fixation or protein overexpression (56)(57)(58). We used correlative cryo- Notably, given that cryo-SIM is much faster than cryo-SMLM and can use a wide variety of spectrally distinct labels, it can be a broadly useful tool in its own right to guide the 3D segmentation of dense FIB-SEM data and ensure the correct identification of specific subcellular features.…”

Section: Correlative Cryo-sr/fib-sem Reveals Vesicle Identities and Tmentioning

confidence: 99%

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Hoffman

Shtengel

et al. 2019

Preprint

119

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words):Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells. modalities (supplementary note 1, table S1), allowing specific molecular components to be visualized at nanoscale resolution in the context of the crowded intracellular environment.However, SR/EM correlation often involves tradeoffs in sample preparation between the retention of fluorescent labels, sufficiently dense heavy metal staining for high contrast EM, and faithful preservation of ultrastructure, particularly when chemical fixation is used (19)(20)(21)(22).Here we describe a pipeline ( fig. S1) for correlative cryo-SR/FIB-SEM imaging of whole cells designed to address these issues. Specifically, cryogenic, as opposed to room temperature, SR performed after high pressure freezing (HPF), allowed us to use a standard EM sample preparation protocol without compromise. We used cryogenic 3D structured illumination (SIM) and single molecule localization (SMLM) microscopy for SR protein specific contrast with 3D FIB-SEM for global contrast of subcellular ultrastructure. The SR modality highlights features not readily apparent from the EM data alone, such as exceptionally long or convoluted endosomes, and permits unique classification of vesicles of like morphology, such as lysosomes, peroxisomes, and mitochondrial-derived vesicles. Cell-wide 3D correlation also reveals unexpected localization patterns of proteins, including intranuclear vesicles positive for an ER marker, intricate web-like structures of adhesion proteins at cell-cell junctions, and heterogeneity in euchromatin or heterochromatin recruitment of transcriptionally-associated histone H3.3 and heterochromatin protein 1α (HP1α) in the nuclei of neural progenitor cells as they transition into differentiated neurons). More generally, whole cell cryo-SR/FIB-SEM can reveal compartmentalized proteins within known subcellular components, help discover new subcellular components, and classify unknown EM morphologies and their roles in cell biology. Cryogenic SR below 10K: motivations and photophysical characterization

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Section: Correlative Cryo-sr/fib-sem Reveals Vesicle Identities and Tmentioning

confidence: 99%

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Hoffman

Shtengel

et al. 2019

Preprint

119

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“…Intravital microscopy has been used to look at the dynamics of renal cell death and could be used for other types of dynamic observation with adapted labelling . Live‐cell correlative light‐electron microscopy (live‐cell CLEM) offers the possibility of simultaneously recording the dynamics of subcellular components whilst imaging their structural properties . Label‐free imaging of live cells has been demonstrated using interferometric scattering microscopy, showing intracellular organelles as well as topological characteristics of the membrane.…”

Section: Discussionmentioning

confidence: 99%

Cellular plasticity: A mechanism for homeostasis in the kidney

et al. 2020

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Cellular plasticity is a topical subject with interest spanning a wide range of fields from developmental biology to regenerative medicine. Even the nomenclature is a subject of debate, and the underlying mechanisms are still under investigation. On top of injury repair, cell plasticity is a constant physiological process in adult organisms and tissues, in response to homeostatic challenges. In this review we discuss two examples of plasticity for the maintenance of homeostasis in the renal systemnamely the renin-producing juxtaglomerular cells (JG cells) and cortical collecting duct (CCD) cells. JG cells show plasticity through recruitment mechanisms, answering the demand for an increase in renin production. In the CCD, cells appear to have the ability to transdifferentiate between principal and intercalated cells to help maintain the highly regulated solute transport levels of that segment. These two cases highlight the complexity of plasticity processes and the role they can play in the kidney. K E Y W O R D Scollecting duct, JG cells, kidney, plasticity, renin 2 of 12 | ASSMUS et Al.

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“…Hela cells were grown on gridded glass coverslips, prepared as described by Fermie et al . 37 . Cells were incubated with fiducial markers at a concentration of 1 μ g/ml dissolved in complete DMEM and incubated for 3 hours, and fixed overnight in 1x PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 , pH = 6.9) containing 4% paraformaldehyde (Sigma) and 0.1% glutaraldehyde (Merck) at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

Fokkema

Fermie

Liv

et al. 2018

Sci Rep

Self Cite

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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.

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

Cited by 85 publications

References 92 publications

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Cellular plasticity: A mechanism for homeostasis in the kidney

Fluorescently Labelled Silica Coated Gold Nanoparticles as Fiducial Markers for Correlative Light and Electron Microscopy

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