Quantification of cell-surface expressed antigenic sites with 5-nm gold markers: Getting close to biologically relevant data

1989 ◽  
Vol 47 ◽  
pp. 1050-1051
Author(s):  
Etienne de Harven ◽  
Hilary Christensen ◽  
Richard Leung ◽  
Cameron Ackerley
Keyword(s):  
Cell Surface ◽  
Human Peripheral Blood ◽  
Contour Length ◽  
Thin Sections ◽  
Gold Particles ◽  
Biologically Relevant ◽  
Transmission Electron ◽  
Entire Cell ◽  
Electron Imaging ◽  
Cell Contour

The T-derived subset of human peripheral blood normal lymphocytes has been selected as a model system to study the usefulness of 5 nm gold markers for quantification of single epitopes expressed on cell surfaces. The chosen epitopes are parts of the CD3 and CD5 molecules and can be specifically identified by hybridoma produced monoclonal antibodies (MoAbs; LEU-4 and LEU-1; Becton-Dick- inson, Mountain view, CA) . An indirect immunolabeling procedure, with goat anti-murine IgG adsorbed on the surface of 5 nm colloidal gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) has been used. Backscattered Electron Imaging (BEI) in a field emission scanning electronmicroscope (SEM) and transmission electron microscopy of thin sections of lymphocytes labeled before plastic embedding, were both used to identify and quantitate gold labeled cell surface sites, Estimating that the thickness of “silver” sections is approximately 60 nm and counting the number of gold particles on the entire cell perimeter, we calculated that, for LEU-4, the number of markers per um2 of cell surface is in the 140-160 range (Fig.l). Cell contour length measurements indicated that the surface of one lymphocyte is approximately 130-160 um2 that of a smooth sphere of identical diameter, reflecting the role of microvilli in expanding the surface area. The total number of gold labeled sites on the surface of one lymphocyte averages, therefore between 20,000 and 24,000 per cell.

scholarly journals Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

1990 ◽  
Vol 38 (3) ◽  
pp. 403-414 ◽  
Author(s):  
A Nanci ◽  
S Zalzal ◽  
C E Smith
Keyword(s):  
Light Microscope ◽  
Cell Structure ◽  
Thin Sections ◽  
Backscattered Electron Imaging ◽  
Cell Compartments ◽  
Tissue Organization ◽  
Transmission Electron ◽  
Backscattered Electron ◽  
Thin Plastic ◽  
Electron Imaging

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.

scholarly journals Caracteres estructurales y ultraestructurales de la gametogénesis de Chara hydropitys (Charophyceae)

2017 ◽  
Vol 65 (4) ◽  
pp. 1507
Author(s):  
Edgar Javier Rincón Barón ◽  
Yenny Magaly Castrillón Bolaños ◽  
Gerardo Andrés Torres ◽  
Fernando Alzate Guarin ◽  
Silvia Espinosa Matías
Keyword(s):  
Electron Microscopy ◽  
Chromatin Condensation ◽  
Side Wall ◽  
Flagellar Apparatus ◽  
Cytoplasmic Inclusions ◽  
Thin Sections ◽  
Transmission Electron ◽  
Structural Details ◽  
Entire Cell ◽  
Ultrathin Sections

In Charophyceae, the oosporangia and antheridia are the respective female and male structures of sexual reproduction. These organs are characterized by their morphological complexity and usefulness in taxonomy and systematics. Here we described the structural and ultraestructural details of Chara hydropitys gametogenesis. The fertile material from the algae was collected in a tributary stream of the Río Meléndez in Cali, Colombia (3º21´23´´N - 76º32´5.2´´W) in March 2011. The specimens were fixed and processed following the standard protocols for inclusion in resin. Thin sections (0.3-0.5 μm) were stained with toluidine O, and were observed by photonic microscopy, and additional ultrathin sections (60-90 nm) were observed by transmission electron microscopy (TEM); other samples were processed and observed by scanning electron microscopy (SEM). We found that the oosporangia are covered with spiral cells, forming 10-12 convolutions and ends in five coronula cells. The immature oosporangia wall is formed by two layers that correspond to the wall of the spiral cells and to the oosphere. In mature stages, the oosporangia wall is composed by six additional layers, three of them are provided by the oosphere and the other three are provided by the spiral cells. Oosphere size increases progressively while the spiral cells grow and divide. The cytoplasm of the immature oosphere does not exhibit conspicuous cytoplasmic inclusions, nevertheless, with the maturation, the number of starch granules increases, occupying most of the cell volume. In the spiral cells of the mature oosporangia we observed large number of chloroplast with starch accumulations, between thylakoid lamellae and a vacuole that occupies almost the entire cell. By using SEM it was possible to appreciate, that the external wall of the oospore, more accurately, on the fossa area, shows verrucose micro-ornamentations with verrucae elevations. In mature antheridia, shield cells are strongly pigmented orange due to the presence of a large number of plastoglobules between thylakoid lamellae. The spermatogenous filaments are developed from cells of the secondary capitulum; those, by unidirectional and sincronic mitotic divisions develop the spermatocytes. The biflagellate antherozoids are developed from the haploid cells by spermiogenesis. The subcellular events related with these division and differentiation processes, include first, chromatin condensation, loss of nucleoli and more activity in dictyosomes. Subsequently, retracts the cytoplasm and the organelles are aligned along the condensed nucleus and flagellar apparatus. Mature antherozoids emerge through a side wall pore of the spermatocytes. All the described events showed that the gametogenesis processes and the gametes structural details in general, are widely conserved in this algae group.

Colloidal Gold Markers: Preparation and Cell Labeling for Transmission and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 530-533
Author(s):  
Robert S. Molday
Keyword(s):  
Cell Surface ◽  
Colloidal Gold ◽  
Critical Evaluation ◽  
Cell Labeling ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Gold Particles ◽  
Backscattered Electrons ◽  
Wide Range ◽  
Scanning Electron

Colloidal gold particles have become one of the most widely used markers to detect, localize and, in some cases, quantitate cell surface and intracellular antigens and receptors since their introduction as transmission electron microscopic (TFM) markers by Faulk and Taylor in 1971 and as scanning electron microscopic (SEM) markers by Horisberger et al. in 1975. This interest in colloidal gold markers for cell labeling is based on their versatile properties for detection under the electron microscope. Colloidal gold particles are highly electron-dense which enables them to be seen under the TEM in thin sections of heavily stained cells. They can be prepared in a wide range of highly uniform sizes for visualization at different magnifications and for multiple labeling studies. Under the SEM, gold particles emit a high quantity of secondary electrons, backscattered electrons and characteristic X-ray signals and as a result, with the appropriate detectors, they can be readily distinguished from cell surface structures having a similar morphological appearance. The successful application of colloidal gold particles as markers for TEM and SEM however requires (i) careful preparation and characterization of both the gold markers and the ligand (protein)-gold conjugates, (ii) utilization of specific labeling techniques employing necessary controls to confirm the specificity of labeling, and (iii) critical evaluation of results in relation to the conditions used in labeling. These aspects of gold labeling will be considered here. Additional information can be obtained from recent reviews dealing specifically with gold markers and more generally with cell labeling techniques.

Characterization of beam-induced thinning and shrinkage of semi-thick sections in the HVEM

1990 ◽  
Vol 48 (3) ◽  
pp. 752-753
Author(s):  
J.R. Kremer ◽  
E.T. O'Toole ◽  
G.P. Wray ◽  
D.M. Mastronarde ◽  
S.J. Mitchell ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Colloidal Gold ◽  
Similar Data ◽  
Thin Sections ◽  
Gold Particles ◽  
Dose Rates ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope

It is well known that irradiation of plastic sections in a conventional transmission electron microscope (cTEM) causes specimen thinning and distortion. Thinning has been observed in the cTEM using several embedding media, using methods such as shrinkage of ordered paracrystalline structures, and shrinkage of sections coated with colloidal gold markers. The total thinning observed in the cTEM (80kev) is 30-50% for thin sections of epon araldite, but similar data do not exist for the HVEM at 1000 kev. Here we describe beam induced thinning and shrinkage of 0.2um sections in the HVEM.Experiments were performed using 0.2um sections of EPOX 812/Araldite or LX112 with 15 nm and 30 nm gold particles affixed to either surface of the section. The sections were initially tilted to approximately 25° and irradiated with known dose rates. Micrographs were taken at different times between 0-20 minutes then the sections were tilted back to 0° for a reference micrograph.

Correlative LM, SEM and TEM of argentaffin cells

1978 ◽  
Vol 36 (2) ◽  
pp. 84-85
Author(s):  
R.G. Frederickson ◽  
D.B. Spagnoli ◽  
P.B. DeNee
Keyword(s):  
Electron Microscopy ◽  
Secondary Electron ◽  
Thin Sections ◽  
Backscattered Electron Imaging ◽  
Selective Staining ◽  
Transmission Electron ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
Different Levels ◽  
Tem Grid

The identification of small numbers of cells containing specific materials or having a particular location in a tissue can be observed cften in light microscopic (IM) sections by selective staining. In contrast, the same cells are often difficult to identify in alternate thin sections on grids by transmission electron microscopy (TEM). Grid bars produce periodic obstructions and consequently prevent the visualization of tissue continuity, making it difficult to identify landmarks. Also, the TEM image of a thin section of tissue does not correlate well with the thicker IM image. This is a matter of interpretation based on the different principles of image formation and the different levels of resolution.The purpose of this paper is to demonstrate a method which allows the same cells identified by IM to be more easily located in thin sections for TEM. This method combines the principles of backscattered electron imaging (BSI), secondary electron imaging (SEI) and heavy metal staining with scanning electron microscopy (SEM).

scholarly journals Evidence for membrane differentiation in polarised leucocytes: the distribution of surface antigens analysed with Ig-gold labelling

1990 ◽  
Vol 95 (3) ◽  
pp. 471-479
Author(s):  
W.S. Haston ◽  
A.F. Maggs
Keyword(s):  
Random Distribution ◽  
Mononuclear Cells ◽  
Surface Antigens ◽  
Membrane Flow ◽  
Thin Sections ◽  
Gold Particles ◽  
Hla Dr ◽  
Transmission Electron ◽  
Morphological Polarity ◽  
Mononuclear Leucocytes

The distribution of a number of leucocyte surface antigens was studied on both round and polarised neutrophil or mononuclear leucocytes using Ig-gold conjugates with transmission electron microscopy. Thin sections of cells, which had been lightly fixed before antibody labelling, were analysed using a statistical method to determine: (1) whether the antigens had a non-random distribution or ‘clustering’ over the cell surface; and (2) whether there was any overall bias in labelling to give a polarised distribution. Comparison between the results of this analysis and cell morphology were made. The results indicated that with the antigens investigated here, CD45, CD15, HLA-DR and CR3, the majority of polarised cells had a calculated direction of overall asymmetry of gold particles that was aligned with the long axis of morphological polarity. Maximal asymmetry was seen in polarised cells labelled for CD45 and HLA-DR, with labelling ratios of up to 6:1 between the front and back of the cell. A number of round mononuclear cells demonstrated significant polarisation of gold particles but this had no apparent morphological correlation and, in general, round cells showed a low degree of asymmetry. However, there was evidence that both round and polarised cells had a non-random distribution or ‘clustering’ of gold particles, which was more marked in morphologically polarised cells and particularly significant in polarised neutrophil leucocytes labelled for CR3. The significance of these results for models of cell locomotion involving membrane flow is discussed.

High-resolution immunogold labeling of cell surfaces: The hypothetical homodimeric nature of the cd5 receptor

1988 ◽  
Vol 46 ◽  
pp. 380-381
Author(s):  
E. de Harven ◽  
H. Christensen
Keyword(s):  
Field Emission ◽  
Molecular Level ◽  
Human Peripheral Blood ◽  
Lanthanum Hexaboride ◽  
Peripheral Blood Lymphocytes ◽  
Cell Surfaces ◽  
Gold Particles ◽  
Human Peripheral Blood Lymphocytes ◽  
Backscattered Electron ◽  
Electron Imaging

Molecules exposed on cell surfaces and labeled with colloidal gold markers can be optimally demonstrated using the backscattered electron imaging (BEI) mode of the scanning electron microscope (SEM). Steric hindrance, however, limits labeling efficiency, making it necessary to use gold markers of small size for labeling at the molecular level. Using a JEOL 840 SEM equipped with a lanthanum hexaboride (LaB6) cathode, 13 nm gold particles were demonstrated. This, however, seems to represent the limit of the resolution of this type of instrument in the BEI mode. Fortunately, it has been demonstrated by Walther and Muller that 5 nm gold particles can be seen in the BEI mode, using field emission SEM.We have confirmed this observation, using the JEOL 890 field emission SEM and a solid state backscattered electron detector. Human peripheral blood lymphocytes prefixed with 0.1% glutaraldehyde, incubated with the murine monoclonal antibody LEU-1 (CD5), were labeled with a goat anti-murine IgG adsorbed on 5 nm gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) according to previously described procedures.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

Studies of Circular DNA Using a New Electron Microscopic Technique

1973 ◽  
Vol 31 ◽  
pp. 596-597
Author(s):  
C. N. Gordon
Keyword(s):  
Electron Microscopy ◽  
Aqueous Salt Solution ◽  
Contour Length ◽  
Cleavage Surface ◽  
Mica Substrate ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Circular Dna ◽  
Transmission Electron ◽  
Electron Microscopic Technique

Gordon and Kleinschmidt have described a new preparative technique for visualizing DNA by electron microscopy. This procedure, which is a modification of Hall's “mica substrate technique”, consists of the following steps: (a) K+ ions on the cleavage surface of native mica are exchanged for Al3+ ions by ion exchange. (b) The mica, with Al3+ in the exchange sites on the surface, is placed in a dilute aqueous salt solution of DNA for several minutes; during this period DNA becomes adsorbed on the surface. (c) The mica with adsorbed DNA is removed from the DNA solution, rinsed, dried and visualized for transmission electron microscopy by Hall's platinum pre-shadow replica technique.In previous studies of circular DNA by this technique, most of the molecules seen were either broken to linears or extensively tangled; in general, it was not possible to obtain suitably large samples of open extended molecules for contour length measurements.

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