scholarly journals Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

2019 ◽  
Author(s):  
Yongdeng Zhang ◽  
Lena K. Schroeder ◽  
Mark D. Lessard ◽  
Phylicia Kidd ◽  
Jeeyun Chung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Super Resolution ◽  
Membrane Structures ◽  
Molecular Specificity ◽  
20 Nm

AbstractCombining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.One Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

Subcellular organization of snRNPS in mammalian cells

1995 ◽  
Vol 53 ◽  
pp. 772-773
Author(s):  
A. Gregory Matera
Keyword(s):  
Rna Processing ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Protein Complexes ◽  
Nuclear Architecture ◽  
Valuable Insight ◽  
Subcellular Organization ◽  
Protein Components ◽  
The Individual

Our laboratory is interested in aspects of eukaryotic gene expression that may be regulated at the level of nuclear architecture and/or localization. Understanding the spatial organization of genes and gene products within mammalian nuclei will provide valuable insight into how the various metabolic processes such as replication, transcription, processing and transport are orchestrated. In order to begin to address these questions, we have chosen to study the cell biology of RNA processing. Small RNA-protein complexes known as ribonucleoproteins (RNPs) are central factors in numerous RNA processing reactions. Much of what is currently known about the subcellular organization of RNPs is based upon the locations of the protein components of RNPs, not the RNA components. Furthermore, since many RNAs share common protein subunits it is essential to develop probes which are specific to the individual RNAs. We utilize highly specific antisense oligonucleotide probes and fluorescence in situ hybridization (FISH) to ascertain the organization of RNPs and their associations with particular subdomains within mammalian cells.

scholarly journals Cell biology of the future: Nanometer-scale cellular cartography

2015 ◽  
Vol 211 (2) ◽  
pp. 211-214 ◽  
Author(s):  
Justin W. Taraska
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Cellular Structure ◽  
Three Dimensional ◽  
Super Resolution ◽  
Molecular Organization ◽  
Nanometer Scale ◽  
Cellular Regulation ◽  
New Developments ◽  
Quantitative Image

Understanding cellular structure is key to understanding cellular regulation. New developments in super-resolution fluorescence imaging, electron microscopy, and quantitative image analysis methods are now providing some of the first three-dimensional dynamic maps of biomolecules at the nanometer scale. These new maps—comprehensive nanometer-scale cellular cartographies—will reveal how the molecular organization of cells influences their diverse and changeable activities.

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

Science ◽  
2020 ◽  
Vol 367 (6475) ◽  
pp. eaaz5357 ◽  
Author(s):  
David P. Hoffman ◽  
Gleb Shtengel ◽  
C. Shan Xu ◽  
Kirby R. Campbell ◽  
Melanie Freeman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Super Resolution ◽  
Whole Cells ◽  
Workflow Optimization ◽  
Associated Proteins ◽  
Block Face

Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam–milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum–associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

scholarly journals Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

1991 ◽  
Vol 39 (11) ◽  
pp. 1495-1506 ◽  
Author(s):  
P M Motte ◽  
R Loppes ◽  
M Menager ◽  
R Deltour
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Spatial Organization ◽  
Higher Plants ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Thin Sections ◽  
Cytoplasmic Dna ◽  
Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

GaN Three Dimensional Nanostructures

1995 ◽  
Vol 395 ◽  
Author(s):  
V. Dmitriev ◽  
K. Irvine ◽  
A. Zubrilov ◽  
D. Tsvetkov ◽  
V. Nikolaev ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Chemical Vapor ◽  
Blue Shift ◽  
High Energy ◽  
Peak Position ◽  
Organic Chemical ◽  
Nanoscale Structures ◽  
Dot Density ◽  
20 Nm

ABSTRACTWe report on the growth and characterization of three dimensional nanoscale structures of GaN. GaN dots were grown by metal organic chemical vapor deposition (MOCVD) on 6H-SiC substrates. The actual size of the dots measured by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) ranged from ∼20 nm to more than 2 μm. The average dot density ranged from 107 to 109 cm−2. The single crystal structure of the dots was verified by reflectance high energy electron diffraction (HEED) and TEM. Cathodoluminescence (CL) and photoluminescence (PL) of the dots were studied at various temperatures and excitation levels. The PL and CL edge peak for the GaN dots exhibited a blue shift as compared with edge peak position for continuous GaN layers grown on SiC.

scholarly journals Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

2010 ◽  
Vol 16 (1) ◽  
pp. 54-63 ◽  
Author(s):  
Niels de Jonge ◽  
Rachid Sougrat ◽  
Brian M. Northan ◽  
Stephen J. Pennycook
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Mammalian Cells ◽  
Three Dimensional ◽  
Axial Resolution ◽  
Beam Radiation ◽  
Thin Sections ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Scanning Transmission

AbstractA three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Frontiers in Cryo Electron Microscopy of Complex Macromolecular Assemblies

2019 ◽  
Vol 21 (1) ◽  
pp. 395-415 ◽  
Author(s):  
Jana Ognjenović ◽  
Reinhard Grisshammer ◽  
Sriram Subramaniam
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Specimen Preparation ◽  
Macromolecular Assemblies ◽  
Cryo Electron Microscopy ◽  
G Protein Coupled ◽  
Improved Methods

In recent years, cryo electron microscopy (cryo-EM) technology has been transformed with the development of better instrumentation, direct electron detectors, improved methods for specimen preparation, and improved software for data analysis. Analyses using single-particle cryo-EM methods have enabled determination of structures of proteins with sizes smaller than 100 kDa and resolutions of ∼2 Å in some cases. The use of electron tomography combined with subvolume averaging is beginning to allow the visualization of macromolecular complexes in their native environment in unprecedented detail. As a result of these advances, solutions to many intractable challenges in structural and cell biology, such as analysis of highly dynamic soluble and membrane-embedded protein complexes or partially ordered protein aggregates, are now within reach. Recent reports of structural studies of G protein–coupled receptors, spliceosomes, and fibrillar specimens illustrate the progress that has been made using cryo-EM methods, and are the main focus of this review.

Electroporation-mediated uptake of proteins into mammalian cells

1990 ◽  
Vol 68 (4) ◽  
pp. 729-734 ◽  
Author(s):  
Helene Lambert ◽  
Roumen Pankov ◽  
Johanne Gauthier ◽  
Ronald Hancock
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Histone H1 ◽  
Dna Transfection ◽  
Optimum Conditions ◽  
Gold Particles ◽  
Protein Uptake ◽  
Particle Uptake ◽  
20 Nm

Proteins of up to 230 kilodaltons are taken up by Chinese hamster ovary fibroblasts exposed to electroporation under conditions generally similar to those used to mediate DNA transfection. The uptake of catalase, ovalbumin, and histone H1 labelled with fluorescein was visualized by fluorescence microscopy. Under the same conditions, the uptake of colloidal gold particles (20 nm diameter) was visualized by electron microscopy. In optimum conditions, about 25% of the cells remained viable and grew normally and about 25% of these retained labelled proteins during two cycles of further growth. About 6 × 104 molecules of catalase were retained per cell. Proteins were taken up when presented to the cells up to 4 h after electroporation, suggesting that mechanisms other than classical electropore formation may operate in these conditions. The proteins were localized in the cytoplasm in a predominantly vesicular pattern and histone H1 entered the nucleus in some cells.Key words: electroporation, protein uptake, gold particle uptake, endocytosis, cell membrane.

scholarly journals Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten W. Tuijtel ◽  
Abraham J. Koster ◽  
Stefan Jakobs ◽  
Frank G. A. Faas ◽  
Thomas H. Sharp
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution
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