Fluoronanogold as a Probe for High Resolution Correlation Between ImmunoFluorescence and Electron Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 476-477
Author(s):  
T. Takizawa ◽  
J. M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Immunofluorescence Microscopy ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Fab Fragment ◽  
Ultrathin Cryosections

[Introduction] Immunocytochemical labeling of cryosections, especially immunofluorescence microscopy using semi-thin (0.5-μm) cryosections, has been a powerful technique for detection of cellular antigens in situ and has been widely employed in cell and molecular biology studies. In many cases, immunofluorescence provides sufficient resolution and sensitivity to answer the question being addressed. However, in certain cases the increased resolution of the electron microscope using ultrathin (90-nm) cryosections may be required to define more precisely the localization of specific molecules. Recently, a unique fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), has been developed for use as a secondary antibody in immunocytochemical applications. It consists of a Fab' fragment of an antibody to which a 1.4-nm gold particle and fluorochromes are conjugated. FNG permits correlative microscopic observation of a sample stained in a single labeling procedure by multiple optical imaging. Recently, we have shown FNG immunocytochemistry on ultrathin cryosections to be valuable for high-resolution correlation of immunofluorescence and immunoelectron microscopy. In the present study, we have examined the utility of FNG as a secondary antibody for immunolabeling of myeloperoxidase (a marker protein for the azurophillic granules) in ultrathin cryosectioned human neutrophils.[Materials and Methods] Purified human neutrophils were fixed with paraformaldehyde, embedded in gelatin, infiltrated with sucrose, cut as ultrathin cryosections, and then collected on formvar film-coated nickel EM grids as described previously. Grids containing ultrathin cryosections were incubated with antimyeloperoxidase and then incubated with FNG.

scholarly journals FluoroNanogold Is a Bifunctional Immunoprobe for Correlative Fluorescence and Electron Microscopy

2000 ◽  
Vol 48 (4) ◽  
pp. 481-485 ◽  
Author(s):  
Toshihiro Takizawa ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
High Spatial Resolution ◽  
Human Neutrophils ◽  
Secondary Antibody ◽  
Marker Protein ◽  
Cytoplasmic Granules ◽  
Fluorescence And Electron Microscopy ◽  
Ultrathin Cryosections

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for highresolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.

In situ High-Resolution Electron Microscopy

1989 ◽  
Vol 47 ◽  
pp. 638-639
Author(s):  
Robert Sinclair
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Thin Foil ◽  
High Resolution Electron Microscopy ◽  
Atomic Scale ◽  
High Electron ◽  
Axis Orientation ◽  
Specimen Holder

The strength of in situ electron microscopy lies in its ability to observe directly material changes which are pertinent to bulk processes. The most rigorous experiments employ a purpose-built specimen holder to simulate specific testing conditions (e.g. heating, cooling, straining, environment), with the associated microstructural changes deduced from appropriate micrographs, diffraction patterns or video-recordings. The influence of the electron microscope itself must always be taken into account (e.g. thin-foil specimen, large electron flux, high electron energy). However when artifacts are overcome, remarkable insight into the mechanisms of the behavior of solids can be achieved. The purpose of this article is to review our work in extending this capability into the high-resolution regime, so that atomic reactions can be followed. In the right circumstances this allows straightforward interpretation of atomic-scale phenomena.Our studies have employed a Philips EM430ST, 300 kV transmission electron microscope (TEM) with about 0.2 nm resolution. To-date we have carried out only heating experiments, utilizing the standard side-entry, single-tilt specimen holder (model number PW 6592), mainly on reactions at semiconductor interfaces. The zone axis orientation necessary for high-resolution TEM is obtained by careful sectioning of the substrate crystals and judicious positioning in the heating holder. The imaging conditions (including drift) can be optimized at a temperature slightly below that required for the changes of interest, so that recording can be initiated immediately on ramping up.

Application of analytical electron microscopy to the study of partitioning of silicon in a 2 wt% Si eutectoid steel

1978 ◽  
Vol 36 (1) ◽  
pp. 616-617
Author(s):  
N. Ridley ◽  
S.A. Al-Salman ◽  
G.W. Lorimer
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Analytical Electron Microscopy ◽  
Eutectoid Steel ◽  
Minimum Diameter ◽  
Thin Foils ◽  
Analytical Electron ◽  
Analytical Electron Microscope ◽  
Transformation Front

The application of the technique of analytical electron microscopy to the study of partitioning of Mn (1) and Cr (2) during the austenite-pearlite transformation in eutectoid steels has been described in previous papers. In both of these investigations, ‘in-situ’ analyses of individual cementite and ferrite plates in thin foils showed that the alloying elements partitioned preferentially to cementite at the transformation front at higher reaction temperatures. At lower temperatures partitioning did not occur and it was possible to identify a ‘no-partition’ temperature for each of the steels examined.In the present work partitioning during the pearlite transformation has been studied in a eutectoid steel containing 1.95 wt% Si. Measurements of pearlite interlamellar spacings showed, however, that except at the highest reaction temperatures the spacing would be too small to make the in-situ analysis of individual cementite plates possible, without interference from adjacent ferrite lamellae. The minimum diameter of the analysis probe on the instrument used, an EMMA-4 analytical electron microscope, was approximately 100 nm.

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.

A Base Specific Single Heavy Atom Stain for Electron Microscopy

1972 ◽  
Vol 30 ◽  
pp. 184-185
Author(s):  
J. P. Langmore ◽  
N. R. Cozzarelli ◽  
A. V. Crewe
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Elastic Scattering ◽  
Heavy Atom ◽  
High Vacuum ◽  
Heavy Atoms ◽  
Large Movement ◽  
Base Sequencing ◽  
Scanning Electron

A system has been developed to allow highly specific derivatization of the thymine bases of DNA with mercurial compounds wich should be visible in the high resolution scanning electron microscope. Three problems must be completely solved before this staining system will be useful for base sequencing by electron microscopy: 1) the staining must be shown to be highly specific for one base, 2) the stained DNA must remain intact in a high vacuum on a thin support film suitable for microscopy, 3) the arrangement of heavy atoms on the DNA must be determined by the elastic scattering of electrons in the microscope without loss or large movement of heavy atoms.

High Resolution Scanning Electron Microscopy

1991 ◽  
Vol 49 ◽  
pp. 478-479
Author(s):  
David Joy ◽  
James Pawley
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Beam ◽  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Electron Probe ◽  
Impact Point ◽  
Interaction Volume ◽  
Scanning Electron

The scanning electron microscope (SEM) builds up an image by sampling contiguous sub-volumes near the surface of the specimen. A fine electron beam selectively excites each sub-volume and then the intensity of some resulting signal is measured. The spatial resolution of images made using such a process is limited by at least three factors. Two of these determine the size of the interaction volume: the size of the electron probe and the extent to which detectable signal is excited from locations remote from the beam impact point. A third limitation emerges from the fact that the probing beam is composed of a finite number of discrete particles and therefore that the accuracy with which any detectable signal can be measured is limited by Poisson statistics applied to this number (or to the number of events actually detected if this is smaller).

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.

Remote On-Line Control of a High-Voltage in situ Transmission Electron Microscope with A Rational User Interface

1996 ◽  
Vol 54 ◽  
pp. 384-385
Author(s):  
M.A. O’Keefe ◽  
J. Taylor ◽  
D. Owen ◽  
B. Crowley ◽  
K.H. Westmacott ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
User Interface ◽  
Transmission Electron Microscope ◽  
High Voltage ◽  
Materials Science ◽  
Transmission Electron ◽  
On Line ◽  
Electron Microscopes

Remote on-line electron microscopy is rapidly becoming more available as improvements continue to be developed in the software and hardware of interfaces and networks. Scanning electron microscopes have been driven remotely across both wide and local area networks. Initial implementations with transmission electron microscopes have targeted unique facilities like an advanced analytical electron microscope, a biological 3-D IVEM and a HVEM capable of in situ materials science applications. As implementations of on-line transmission electron microscopy become more widespread, it is essential that suitable standards be developed and followed. Two such standards have been proposed for a high-level protocol language for on-line access, and we have proposed a rational graphical user interface. The user interface we present here is based on experience gained with a full-function materials science application providing users of the National Center for Electron Microscopy with remote on-line access to a 1.5MeV Kratos EM-1500 in situ high-voltage transmission electron microscope via existing wide area networks. We have developed and implemented, and are continuing to refine, a set of tools, protocols, and interfaces to run the Kratos EM-1500 on-line for collaborative research. Computer tools for capturing and manipulating real-time video signals are integrated into a standardized user interface that may be used for remote access to any transmission electron microscope equipped with a suitable control computer.

High-Resolution Resistance Mapping in SEM

2018 ◽  
Author(s):  
Grigore Moldovan ◽  
Wolfgang Joachimi ◽  
Guillaume Boetsch ◽  
Jörg Jatzkowski ◽  
Frank Altman
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Reference Structure ◽  
Total Resistance ◽  
Embedded Electronics ◽  
Improved Performance ◽  
Mapping Techniques ◽  
Scanning Electron

Abstract This work presents advanced resistance mapping techniques based on Scanning Electron Microscopy (SEM) with nanoprobing systems and the related embedded electronics. Focus is placed on recent advances to reduce noise and increase speed, such as integration of dedicated in situ electronics into the nanoprobing platform, as well as an important transition from current-sensitive to voltagesensitive amplification. We show that it is now possible to record resistance maps with a resistance sensitivity in the 10W range, even when the total resistance of the mapped structures is in the range of 100W. A reference structure is used to illustrate the improved performance, and a lowresistance failure case is presented as an example of analysis made possible by these developments.

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