2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

Flechsler, Jennifer; Heimerl, Thomas; Pickl, Carolin; Rachel, Reinhard; Stierhof, York‐Dieter; Klingl, Andreas

doi:10.1002/jemt.23459

Cited by 11 publications

(11 citation statements)

References 76 publications

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“…The cells were concentrated by centrifugation and the cells were high-pressure frozen using a Leica HPM100 (Leica Microsystems, Wetzlar, Germany). This was followed by freeze-substitution with 0.2% osmium tetroxide, 0.1% uranyl acetate, 9.3% water in water-free acetone in a Leica AFS 2 (Leica Microsystems, Wetzlar, Germany) as described previously (Flechsler et al , 2020). After embedding in Epon 812 substitute resin (Fluka Chemie AG, Buchs Switzerland), the cells were ultrathin sectioned (50 to 100 nm thickness) and post-stained for 1 min with lead citrate.…”

Section: Methodsmentioning

confidence: 99%

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Janda

Ludwig²,

Rybak³

et al. 2021

Preprint

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Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

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Section: Methodsmentioning

confidence: 99%

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Janda

Ludwig²,

Rybak³

et al. 2021

Preprint

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“…FIB-SEM and serial sectioning-TEM were applied successfully to image the well-preserved ultrastructure of neuronal networks at high resolution ( Figures 5 , 6 ). For future studies, the ultrathin sections prepared by serial ultramicrotome sectioning can be combined with immunolabeling to allow the localization of macromolecules at high resolution in the cellular context ( Lucocq, 1994 ; Schwarz and Humbel, 2007 ; Flechsler et al, 2020 ). Theoretically, another approach known as serial block-face scanning electron microscopy (SBF-SEM) would enable the imaging and investigation of neurites inside the microchannels with a relatively larger field of view as compared to FIB-SEM.…”

Section: Discussionmentioning

confidence: 99%

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging

et al. 2021

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The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam – scanning electron microscopy (FIB-SEM) and serial sectioning – transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

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“…Thus, it has to be applied when samples are already immunogold labeled. In some post-embedding immunogold techniques, osmium was used and good labeling still had been achieved (Flechsler et al, 2020).…”

Section: Stainingmentioning

confidence: 99%

Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles

Ivanchenko

Indzhykulian

Corey

2021

Front. Cell Dev. Biol.

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Hair cells—the sensory cells of the vertebrate inner ear—bear at their apical surfaces a bundle of actin-filled protrusions called stereocilia, which mediate the cells’ mechanosensitivity. Hereditary deafness is often associated with morphological disorganization of stereocilia bundles, with the absence or mislocalization within stereocilia of specific proteins. Thus, stereocilia bundles are closely examined to understand most animal models of hereditary hearing loss. Because stereocilia have a diameter less than a wavelength of light, light microscopy is not adequate to reveal subtle changes in morphology or protein localization. Instead, electron microscopy (EM) has proven essential for understanding stereocilia bundle development, maintenance, normal function, and dysfunction in disease. Here we review a set of EM imaging techniques commonly used to study stereocilia, including optimal sample preparation and best imaging practices. These include conventional and immunogold transmission electron microscopy (TEM) and scanning electron microscopy (SEM), as well as focused-ion-beam scanning electron microscopy (FIB-SEM), which enables 3-D serial reconstruction of resin-embedded biological structures at a resolution of a few nanometers. Parameters for optimal sample preparation, fixation, immunogold labeling, metal coating and imaging are discussed. Special attention is given to protein localization in stereocilia using immunogold labeling. Finally, we describe the advantages and limitations of these EM techniques and their suitability for different types of studies.

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2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

Cited by 11 publications

References 76 publications

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

A Compartmentalized Neuronal Cell-Culture Platform Compatible With Cryo-Fixation by High-Pressure Freezing for Ultrastructural Imaging

Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles

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