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.

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.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

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.

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

Techniques Involved in the Acquisition of Serial Sections of the Atrioventricular Node for Light and Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 226-227
Author(s):  
Sheila S. Emmett ◽  
J. C. Thaemert
Keyword(s):  
Electron Microscopy ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
Atrioventricular Node ◽  
Electron Microscopic Level ◽  
Serial Sections ◽  
Electron Microscopic ◽  
Trapezoidal Shape ◽  
Old Mice

The acquisition of serial sections of the atrioventricular node for light and electron microscopy is a formidable task. Ordinary techniques are not adequate if the best possible results are to be achieved at the electron microscopic level. The techniques outlined below have proven to be valuable in locating and determining the position of the AV node.Whole hearts of 2-week old mice were fixed, in situ, by perfusion with 1% phosphate-buffered osmium tetroxide. The hearts were removed from the animals, sectioned transversely into 3 slices approximately equal in thickness, dehydrated in graded concentrations of ethanol and embedded in Epon 812. The block faces were trimmed to a trapezoidal shape ranging in size from 0.75 x 1 mm to 4 x 5 mm. Serial sections approximately 2 microns in thickness were cut with glass knives on a Porter-Blum MT-2 Ultramicrotome. While floating on a drop of water on the knife, each section was stretched with 1 drop of a 1:1, xylene in chloroform mixture applied directly to the section. The sections were picked up individually with a brush, transferred to a glass slide and oven dried for several hours prior to staining.

scholarly journals X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

2014 ◽  
Vol 21 (1) ◽  
pp. 231-238 ◽  
Author(s):  
Eric A. Bushong ◽  
Donald D. Johnson ◽  
Keun-Young Kim ◽  
Masako Terada ◽  
Megumi Hatori ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Microscopy Study ◽  
Electron Microscopic ◽  
Intense Staining ◽  
X Ray ◽  
Block Face ◽  
Scanning Electron

AbstractThe recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

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