scholarly journals Analysis of Antiphotobleaching Reagents for use with FluoroNanogold in Correlative Microscopy

2000 ◽  
Vol 48 (3) ◽  
pp. 433-436 ◽  
Author(s):  
Toshihiro Takizawa ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Detection System ◽  
Gold Cluster ◽  
Photochemical Reactions ◽  
The Other ◽  
Cluster Compound ◽  
Fluorescence Signal ◽  
Correlative Microscopy ◽  
Important Approach ◽  
Covalently Linked

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO], p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.

scholarly journals Efficient Immunocytochemical Labeling of Leukocyte Microtubules with FluoroNanogold: An Important Tool for Correlative Microscopy

1997 ◽  
Vol 45 (5) ◽  
pp. 631-642 ◽  
Author(s):  
John M. Robinson ◽  
Dale D. Vandré
Keyword(s):  
Electron Microscopy ◽  
Fluorescence Microscopy ◽  
Gold Particle ◽  
Model System ◽  
The Other ◽  
Secondary Antibody ◽  
Correlative Microscopy ◽  
Silver Enhancement ◽  
Fab Fragment ◽  
Gold Particles

We tested the immunoprobe FluoroNanogold (FNG) for its utility as an immunocytochemical labeling reagent. This immunoprobe consists of a 1.4-nm gold particle to which a specific Fab' fragment and a fluorochrome are conjugated. We employed the microtubules (MTs) of human phagocytic leukocytes as a model system for testing the usefulness of FNG as a secondary antibody for immunocytochemistry. We show that these fluorescently labeled ultrasmall immunogold particles are very efficient for labeling MTs in these cells. The signal from FNG can be detected directly by fluorescence microscopy or indirectly by other modes of optical microscopy and electron microscopy, after silver-enhancement of the gold. The spatial resolution of immunolabeled MTs obtained with FNG and silver enhancement was comparable to that of conventional immunofluorescence detection. Colloidal gold (5-nm and 10-nm in diameter), on the other hand, failed to label MTs in cells prepared in a similar manner. This difference in labeling was due in large part to greater penetration of 1.4-nm gold into aldehyde-fixed cells than either 5-nm or 10-nm gold particles. The fluorescent 1.4-nm immunoprobe was shown to be an important new tool for general use in correlative microscopy.

scholarly journals Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle

1998 ◽  
Vol 46 (10) ◽  
pp. 1097-1102 ◽  
Author(s):  
Toshihiro Takizawa ◽  
Kouki Suzuki ◽  
John M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Imaging Techniques ◽  
Human Neutrophils ◽  
Fluorescence Signal ◽  
Correlative Microscopy ◽  
Reporter System ◽  
Fluorescence And Electron Microscopy ◽  
Specific Granules ◽  
Ultrathin Cryosections ◽  
Proof Of Principle

We demonstrate a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to be a versatile reporter system for immunocytochemical labeling of ultrathin cryosections. FNG-labeled molecules in the same ultrathin cryosections can be resolved by two imaging techniques (i.e., fluorescence and electron microscopy). Lactoferrin, a marker protein for the specific granules in human neutrophils, was employed as the target for FNG immunolabeling. The spatial resolution of the fluorescence signal from FNG-labeled specific granules was compatible with that of silver-enhanced gold signal from the same granules in electron microscopy. Our results confirm that FNG can be used as a probe for highresolution correlation between immunofluorescence and electron microscopy.

scholarly journals Two improved methods for preparing ferritin-protein conjugates for electron microscopy.

1975 ◽  
Vol 64 (2) ◽  
pp. 331-339 ◽  
Author(s):  
Y Kishida ◽  
B R Olsen ◽  
R A Berg ◽  
D J Prockop
Keyword(s):  
Electron Microscopy ◽  
Water Soluble ◽  
The Other ◽  
Activated Esters ◽  
Protein Conjugates ◽  
Antibody Conjugates ◽  
Covalently Linked ◽  
Improved Methods ◽  
In Cells

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin-antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.

Development of 200 kV Scanning-Transmission Electron Microscope

1974 ◽  
Vol 32 ◽  
pp. 410-411
Author(s):  
H. Koike ◽  
S. Sakurai ◽  
K. Ueno ◽  
M. Watanabe
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Transmission Electron Microscope ◽  
The Other ◽  
Morphological Observation ◽  
Magnetic Domains ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Backscattered Electron ◽  
Increasing Demand

In recent years, there has been increasing demand for higher voltage SEMs, in the field of surface observation, especially that of magnetic domains, dislocations, and electron channeling patterns by backscattered electron microscopy. On the other hand, the resolution of the CTEM has now reached 1 ∼ 2Å, and several reports have recently been made on the observation of atom images, indicating that the ultimate goal of morphological observation has beem nearly achieved.

Cryomicroscopy of multiphase latices

1991 ◽  
Vol 49 ◽  
pp. 1060-1061
Author(s):  
O. L. Shaffer ◽  
M.S. El-Aasser ◽  
C. L. Zhao ◽  
M. A. Winnik ◽  
R. R. Shivers
Keyword(s):  
Electron Microscopy ◽  
Radiation Damage ◽  
Freeze Fracture ◽  
Particle Morphology ◽  
Second Stage ◽  
Transmission Electron ◽  
Important Approach ◽  
Carbon Coated ◽  
Preparation Techniques

Transmission electron microscopy is an important approach to the characterization of the morphology of multiphase latices. Various sample preparation techniques have been applied to multiphase latices such as OsO4, RuO4 and CsOH stains to distinguish the polymer phases or domains. Radiation damage by an electron beam of latices imbedded in ice has also been used as a technique to study particle morphology. Further studies have been developed in the use of freeze-fracture and the effect of differential radiation damage at liquid nitrogen temperatures of the latex particles embedded in ice and not embedded.Two different series of two-stage latices were prepared with (1) a poly(methyl methacrylate) (PMMA) seed and poly(styrene) (PS) second stage; (2) a PS seed and PMMA second stage. Both series have varying amounts of second-stage monomer which was added to the seed latex semicontinuously. A drop of diluted latex was placed on a 200-mesh Formvar-carbon coated copper grid.

Comparative strengths and weaknesses of peroxidase and colloidal gold in pre- and post-embedding immunolabeling techniques

1987 ◽  
Vol 45 ◽  
pp. 1002-1005
Author(s):  
D. R. Abrahamson ◽  
P. L. St.John ◽  
E. W. Perry
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Light Microscopy ◽  
Colloidal Gold ◽  
Light Microscope ◽  
Ultrastructural Localization ◽  
The Other ◽  
Quantitative Studies ◽  
Dense Suspension ◽  
Antigen Localization

Antibodies coupled to tracers for electron microscopy have been instrumental in the ultrastructural localization of antigens within cells and tissues. Among the most popular tracers are horseradish peroxidase (HRP), an enzyme that yields an osmiophilic reaction product, and colloidal gold, an electron dense suspension of particles. Some advantages of IgG-HRP conjugates are that they are readily synthesized, relatively small, and the immunolabeling obtained in a given experiment can be evaluated in the light microscope. In contrast, colloidal gold conjugates are available in different size ranges and multiple labeling as well as quantitative studies can therefore be undertaken through particle counting. On the other hand, gold conjugates are generally larger than those of HRP but usually can not be visualized with light microscopy. Concern has been raised, however, that HRP reaction product, which is exquisitely sensitive when generated properly, may in some cases distribute to sites distant from the original binding of the conjugate and therefore result in spurious antigen localization.

Life Beyond 1MeV

1980 ◽  
Vol 38 ◽  
pp. 30-33
Author(s):  
K.H. Westmacott
Keyword(s):  
Electron Microscopy ◽  
Past Experience ◽  
The Other ◽  
Good Case ◽  
Other Hand ◽  
The U.S

Life beyond 1MeV – like life after 40 – is not too different unless one takes advantage of past experience and is receptive to new opportunities. At first glance, the returns on performing electron microscopy at voltages greater than 1MeV diminish rather rapidly as the curves which describe the well-known advantages of HVEM often tend towards saturation. However, in a country with a significant HVEM capability, a good case can be made for investing in instruments with a range of maximum accelerating voltages. In this regard, the 1.5MeV KRATOS HVEM being installed in Berkeley will complement the other 650KeV, 1MeV, and 1.2MeV instruments currently operating in the U.S. One other consideration suggests that 1.5MeV is an optimum voltage machine – Its additional advantages may be purchased for not much more than a 1MeV instrument. On the other hand, the 3MeV HVEM's which seem to be operated at 2MeV maximum, are much more expensive.

Normal Synaptonemal Complex and Abnormal Recombination Nodules in Two Alleles of the Drosophila Meiotic Mutant mei-W68

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1337-1356 ◽  
Author(s):  
Adelaide T C Carpenter
Keyword(s):  
Electron Microscopy ◽  
Synaptonemal Complex ◽  
High Frequency ◽  
Serial Section ◽  
The Other ◽  
Wild Type ◽  
Recombination Nodules ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy

Abstract The meiotic phenotypes of two mutant alleles of the mei-W68 gene, 1 and L1, were studied by genetics and by serial-section electron microscopy. Despite no or reduced exchange, both mutant alleles have normal synaptonemal complex. However, neither has any early recombination nodules; instead, both exhibit high numbers of very long (up to 2 μm) structures here named “noodles.” These are hypothesized to be formed by the unchecked extension of identical but much shorter structures ephemerally seen in wild type, which may be precursors of early recombination nodules. Although the mei-W68L1 allele is identical to the mei-W681 allele in both the absence of early recombination nodules and a high frequency of noodles (i.e., it is amorphic for the noodle phene), it is hypomorphic in its effects on exchange and late recombination nodules. The differential effects of this allele on early and late recombination nodules are consistent with the hypothesis that Drosophila females have two separate recombination pathways—one for simple gene conversion, the other for exchange.

Use of electron microscopy to characterize the surfaces of flocculent and nonflocculent yeast cells

1989 ◽  
Vol 35 (12) ◽  
pp. 1081-1086 ◽  
Author(s):  
Byron F. Johnson ◽  
L. C. Sowden ◽  
Teena Walker ◽  
Bong Y. Yoo ◽  
Gode B. Calleja
Keyword(s):  
Electron Microscopy ◽  
Fission Yeast ◽  
Budding Yeast ◽  
The Other ◽  
Yeast Cells ◽  
Scanning Electron Micrographs ◽  
Soft Or ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Scanning Electron

The surfaces of flocculent and nonflocculent yeast cells have been examined by electron microscopy. Nonextractive preparative procedures for scanning electron microscopy allow comparison in which sharp or softened images of surface details (scars, etc.) are the criteria for relative abundance of flocculum material. Asexually flocculent budding-yeast cells cannot be distinguished from nonflocculent budding-yeast cells in scanning electron micrographs because the scar details of both are well resolved, being hard and sharp. On the other hand, flocculent fission-yeast cells are readily distinguished from nonflocculent cells because fission scars are mostly soft or obscured on flocculent cells, but sharp on nonflocculent cells. Sexually and asexually flocculent fission-yeast cells cannot be distinguished from one another as both are heavily clad in "mucilaginous" or "hairy" coverings. Examination of lightly extracted and heavily extracted flocculent fission-yeast cells by transmission electron microscopy provides micrographs consistent with the scanning electron micrographs.Key words: flocculation, budding yeast, fission yeast, scanning, transmission.

Sporogenesis and septum schizolysis in Dipodascus aggregates

1989 ◽  
Vol 67 (7) ◽  
pp. 2150-2153 ◽  
Author(s):  
A. Tsuneda ◽  
S. Murakami
Keyword(s):  
Electron Microscopy ◽  
Freeze Drying ◽  
The Other ◽  
Cross Wall

Asci, ascospores, and arthroconidia of Dipodascus aggregatus were examined by electron microscopy. Freeze-drying of mature asci caused rupture of the ascal walls and made it possible to observe capsulated ascospores in situ. Two types of septa occurred: (i) one having a remarkably thickened cross wall which protruded to form a circumferential ridge on the hyphae, and (ii) the other without such a bulging ridge. Schizolysis of these septa resulted in the formation of morphologically distinct arthroconidia. The scizolytic process of the ridged septa was demonstrated in detail. There was no evidence to support that the plasmadesmal canals in the septa function as disjunctive pegs in the process of arthroconidial separation.

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