Chiral Os(II) Polypyridyl Complexes as Enantioselective Nuclear DNA Imaging Agents Especially Suitable for Correlative High-Resolution Light and Electron Microscopy Studies

2020 ◽  
Vol 12 (3) ◽  
pp. 3465-3473 ◽  
Author(s):  
Rong Huang ◽  
Feng-Ping Feng ◽  
Chun-Hua Huang ◽  
Li Mao ◽  
Miao Tang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nuclear Dna ◽  
Light And Electron Microscopy ◽  
Imaging Agents ◽  
Polypyridyl Complexes ◽  
Dna Imaging

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Low-Voltage, High-Resolution, Scanning Electron Microscopy of Platelet-Bound Fibrinogen

1996 ◽  
Vol 54 ◽  
pp. 316-317
Author(s):  
S.R. Simmons ◽  
R.M. Albrecht
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Low Voltage ◽  
Blood Clot ◽  
Light And Electron Microscopy ◽  
Essential Element ◽  
Specimen Preparation ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Adhesive Interactions

An essential element in blood clot formation is fibrinogen-mediated platelet aggregation. Fibrinogen is an adhesive plasma protein which binds to the αIIbp3 integrin on activated platelet surfaces. Platelets do not aggregate in the absence of fibrinogen binding, and fibrinogen bound to surfaces of platelets in aggregates is localized to regions of platelet-platelet contact. The fibrinogen molecule is symmetrical and bifunctional and may directly bridge the gap between platelets to bind to receptors on two adjacent platelets. However, the precise mechanism by which fibrinogen links platelets is unclear.Previously we have utilized colloidal gold labeling with correlative light and electron microscopy to investigate the binding of fibrinogen to receptors on surfaces of spread, substrate adherent platelets. The initial binding of gold-conjugated fibrinogen (FgnAu) and subsequent ligand-triggered receptor movement was followed on living platelets by video-enhanced light microscopy. Fibrinogen receptors initially are dispersed over much of the platelet surface and move centripetally upon fibrinogen binding, ultimately forming a band of bound fibrinogen on the platelet surface overlying a densely woven band of actin filaments surrounding the central granulomere. After preparation for electron microscopy, the same platelets as were followed in the light microscope were located in the high voltage TEM and the low voltage, high resolution, SEM (Hitachi S-900) and the final locations of the gold labeled receptor/ligand complexes were determined relative to internal or surface ultrastructure, respectively. More recently, we have utilized the SEM operated at low (1-2 kV) beam voltage to examine in detail the binding of unlabeled fibrinogen to platelets. With appropriate specimen preparation, individual cell surface macromolecules can be resolved in situ by low voltage SEM. In addition to the centripetal receptor redistribution seen with FgnAu, unlabeled fibrinogen appeared to undergo self-adhesive interactions following binding to platelet fibrinogen receptors, forming small, branched and globular protein aggregates during translocation across the platelet surface.(Fig. 1)

scholarly journals Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

scholarly journals Correlative Organelle Microscopy: fluorescence guided volume electron microscopy of intracellular processes

2021 ◽  
Author(s):  
Sergey Loginov ◽  
Job Fermie ◽  
Jantina Fokkema ◽  
Alexandra V Agronskaia ◽  
Cecilia de Heus ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Temporal Organization ◽  
Data Sets ◽  
3 Dimensional ◽  
Cellular Physiology ◽  
3D Fluorescence Microscopy

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (<100nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) data sets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated in a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume-EM, and obviates the need for post correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.

Metal-Polymer Hybrid Nanoparticles for Correlative High-Resolution Light and Electron Microscopy

2017 ◽  
Vol 34 (10) ◽  
pp. 1700180 ◽  
Author(s):  
Martin Reifarth ◽  
Elisabeth Preußger ◽  
Ulrich S. Schubert ◽  
Rainer Heintzmann ◽  
Stephanie Hoeppener
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Hybrid Nanoparticles ◽  
Polymer Hybrid ◽  
Metal Polymer

scholarly journals Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Pieter Baatsen ◽  
Sergio Gabarre ◽  
Katlijn Vints ◽  
Rosanne Wouters ◽  
Dorien Vandael ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Fluorescent Proteins ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
High Resolution Electron Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Tissue Samples ◽  
Rapid Acquisition

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Feulgen-thiocarbohydrazide-silver methenamine(FTS) reaction: An improved silver methodology for light and electron microscopy of DNA

1984 ◽  
Vol 42 ◽  
pp. 262-263 ◽  
Author(s):  
J. S. Hanker ◽  
B. L. Giammara
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Molecular Species ◽  
Light And Electron Microscopy ◽  
Ultrastructural Level ◽  
Feulgen Reaction ◽  
Full Paper ◽  
End Products ◽  
Silver Compounds

A number of cytochemical methods have been Introduced to reveal nuclear components at the ultrastructural level. Many of these, however, are topographic, i.e., identify structures rather than molecular species (1). Indeed, even the search for modifications of the Feulgen reaction to render the nuclear sites of chromatin or DNA electron opaque have not met with a great deal of success (1). Thus the aldehyde or pseudoaldehyde groups on apurinic acids, generated by the action of IN HCl at 60° on DNA, could not be shown by condensation with thiocarbohydrazide (TCH) or thiosemicarbazide and subsequent osmication (2,3). Feulgen reactions with silver end products resulting from the reaction of silver compounds with apurinic-acids have been used since 1924, but these reactions either lack specificity or cannot be used for high resolution studies. Although an abstract appeared in 1972 on a Feulgen reaction employing TCH and silver proteinate (4), neither light micrographs nor a full paper have appeared.

Tannin-ferrocyanide OsO4 method for scanning electron microscopy with use of microwave irradiation

1990 ◽  
Vol 48 (3) ◽  
pp. 26-27
Author(s):  
Gen Takahashi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Microwave Irradiation ◽  
Heat Generation ◽  
Light And Electron Microscopy ◽  
Staining Methods ◽  
Metal Decoration ◽  
Time Required ◽  
Scanning Electron

The disadvantages of metal-coating techniques in high resolution SEM are limitation of resolution due to the thickness of coating, metal decoration of the surface ultrastructures, the lack of uniformity and continuity of coating or specimen damage during coating due to heat generation. In order to overcome these disadvantages and to obtain the better ultrastructural preservation after drying of specimens, the conductive staining methods have been devised.˜4The microwave irradiation(MWI) has recently been used for fixation of biological specimens for light and electron microscopy, immunohistochemistry and for acceleration of the time required for tissue processing and staining. In the present study, MWI can be successfully applied to each step in the tannin- ferrocyanide-OsO4(TA- FeCN-Os) method for high resolution SEM.The TA- FeCNsOs method for high resolution SEM [A] Primary Osmication(Fixation): double fixation with glutaraldehyde-paraformaldehyde and OsO4, TA-FeCN-Os method for TEM,Osmium-DMSO-Osmium, prolonged osmication or osmication after extraction with saponin, glycerol or detergents.

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