Browse
Submit Data
Databases
API
Help

Dataset Information

11 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A robust method for particulate detection of a genetic tag for 3D electron microscopy.

ABSTRACT: Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

SUBMITTER: Rae J

PROVIDER: S-EPMC8104959 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

High data output and automated 3D correlative light-electron microscopy method.

Project description:Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision-accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.

| S-EPMC2635477 | biostudies-literature

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Project description:Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.

| S-EPMC7238333 | biostudies-literature

Spatial Transcriptomics correlated Electron Microscopy

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-06-21 | GSE202638 | GEO

Robust Cell Detection for Large-Scale 3D Microscopy Using GPU-Accelerated Iterative Voting.

Project description:High-throughput imaging techniques, such as Knife-Edge Scanning Microscopy (KESM),are capable of acquiring three-dimensional whole-organ images at sub-micrometer resolution. These images are challenging to segment since they can exceed several terabytes (TB) in size, requiring extremely fast and fully automated algorithms. Staining techniques are limited to contrast agents that can be applied to large samples and imaged in a single pass. This requires maximizing the number of structures labeled in a single channel, resulting in images that are densely packed with spatial features. In this paper, we propose a three-dimensional approach for locating cells based on iterative voting. Due to the computational complexity of this algorithm, a highly efficient GPU implementation is required to make it practical on large data sets. The proposed algorithm has a limited number of input parameters and is highly parallel.

| S-EPMC5932171 | biostudies-literature

Spatial Transcriptomics correlated Electron Microscopy [MERFISH]

Project description:Current spatial transcriptomics methods provide molecular and spatial information but no morphological readout. Here, we present STEM - a method that correlates multiplexed error-robust FISH with electron microscopy from neighboring tissue sections of the same sample. STEM links transcriptional and spatial organization of single cells with ultrastructural morphology of the tissue in vivo. Using STEM to characterize demyelinated white-matter lesions allowed us to link morphology of myelin-laden foamy microglia to transcriptional signature. Moreover, we revealed that interferon-response microglia have unique morphology and are enriched near CD8 T-cells.

2023-06-21 | GSE202623 | GEO

Spatial Transcriptomics correlated Electron Microscopy [scRNA-Seq]

Project description:Current spatial transcriptomics methods identify cell types and states in a spatial context but lack morphological information. Electron microscopy, in contrast, provides structural details at nanometer resolution without decoding the diverse cellular states and identity. STEM address this limitation by correlating multiplexed error-robust FISH with electron microscopy from adjacent tissue sections. Using STEM to characterize demyelinated lesions in mice, we were able to bridge spatially resolved transcriptional data with morphological information on cell identities. This approach allowed us to link the morphology of foamy microglia and interferon-response microglia with their transcriptional signatures.

2023-06-21 | GSE202636 | GEO

Electron Microscopy for 3D Scaffolds-Cell Biointerface Characterization.

Project description:Cell fate is largely determined by interactions that occur at the interface between cells and their surrounding microenvironment. For this reason, especially in the field of tissue-engineering, there is a growing interest in developing techniques that allow evaluating cell-material interaction at the nanoscale, particularly focusing on cell adhesion processes. While for 2D culturing systems a consolidated series of tools already satisfy this need, in 3D environments, more closely recapitulating complex in vivo structures, there is still a lack of procedures furthering the comprehension of cell-material interactions. Here, the use of scanning electron microscopy coupled with a focused ion beam (SEM/FIB) for the characterization of cell interactions with 3D scaffolds obtained by different fabrication techniques is reported for the first time. The results clearly show the capability of the developed approach to preserve and finely resolve scaffold-cell interfaces highlighting details such as plasma membrane arrangement, extracellular matrix architecture and composition, and cellular structures playing a role in cell adhesion to the surface. It is anticipated that the developed approach will be relevant for the design of efficient cell-instructive platforms in the study of cellular guidance strategies for tissue-engineering applications as well as for in vitro 3D models.

| S-EPMC7340848 | biostudies-literature

Navigating 3D electron microscopy maps with EM-SURFER.

Project description:The Electron Microscopy DataBank (EMDB) is growing rapidly, accumulating biological structural data obtained mainly by electron microscopy and tomography, which are emerging techniques for determining large biomolecular complex and subcellular structures. Together with the Protein Data Bank (PDB), EMDB is becoming a fundamental resource of the tertiary structures of biological macromolecules. To take full advantage of this indispensable resource, the ability to search the database by structural similarity is essential. However, unlike high-resolution structures stored in PDB, methods for comparing low-resolution electron microscopy (EM) density maps in EMDB are not well established.We developed a computational method for efficiently searching low-resolution EM maps. The method uses a compact fingerprint representation of EM maps based on the 3D Zernike descriptor, which is derived from a mathematical series expansion for EM maps that are considered as 3D functions. The method is implemented in a web server named EM-SURFER, which allows users to search against the entire EMDB in real-time. EM-SURFER compares the global shapes of EM maps. Examples of search results from different types of query structures are discussed.We developed EM-SURFER, which retrieves structurally relevant matches for query EM maps from EMDB within seconds. The unique capability of EM-SURFER to detect 3D shape similarity of low-resolution EM maps should prove invaluable in structural biology.

| S-EPMC4448178 | biostudies-literature

VIPER is a genetically encoded peptide tag for fluorescence and electron microscopy.

Project description:Many discoveries in cell biology rely on making specific proteins visible within their native cellular environment. There are various genetically encoded tags, such as fluorescent proteins, developed for fluorescence microscopy (FM). However, there are almost no genetically encoded tags that enable cellular proteins to be observed by both FM and electron microscopy (EM). Herein, we describe a technology for labeling proteins with diverse chemical reporters, including bright organic fluorophores for FM and electron-dense nanoparticles for EM. Our technology uses versatile interacting peptide (VIP) tags, a class of genetically encoded tag. We present VIPER, which consists of a coiled-coil heterodimer formed between the genetic tag, CoilE, and a probe-labeled peptide, CoilR. Using confocal FM, we demonstrate that VIPER can be used to highlight subcellular structures or to image receptor-mediated iron uptake. Additionally, we used VIPER to image the iron uptake machinery by correlative light and EM (CLEM). VIPER compared favorably with immunolabeling for imaging proteins by CLEM, and is an enabling technology for protein targets that cannot be immunolabeled. VIPER is a versatile peptide tag that can be used to label and track proteins with diverse chemical reporters observable by both FM and EM instrumentation.

| S-EPMC6304991 | biostudies-literature

Robust ultraclean atomically thin membranes for atomic-resolution electron microscopy.

Project description:The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.

| S-EPMC6987160 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data