Application of Backscattered Electron Imaging to the Study of Scavenger Receptor-Mediated Endocytosis by Sinusoidal Cells in Bone Marrow

1985 ◽  
Vol 43 ◽  
pp. 542-545
Author(s):  
R.P. Becker ◽  
J.S. Geoffroy
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Coated Pits ◽  
Thin Sections ◽  
Backscattered Electron Imaging ◽  
Sinusoidal Cells ◽  
Dense Bodies ◽  
Sinusoidal Cell ◽  
Backscattered Electron ◽  
Electron Imaging

The endothelial cells lining the postcapillary venous sinuses (sinusoids) in bone marrow take up colloidal gold-bovine serum albumin (BSA-Au) conjugates by means of a pathway involving coated pits and vesicles. Endocytosis of BSA- Au by these sinusoidal endothelial cells (sinusoidal cells) is rapid. Within one minute of pulse presentation (5 sec; intraaortic injection) with BSA-Au the probe is internalized and processed through pleomorphic endosomes to dense bodies known to be secondary lysosomes. By this time, 17% of the sinusoidal cell related BSA-Au is associated with the surface, while 83% is internalized, of which 2% is present in lysosomes. By four minutes, less than 8% of the observed BSA-Au is not internalized, the bulk being present predominantly in large pleomorphic vacuoles and dense bodies.That the endocytic process involves coated pits and vesicles prompts the suggestion that it may be receptor mediated. In order to investigate this possibility, biochemical and morphological studies were performed to determine the specificity and saturability of the putative receptor. Morphological analysis of TEM thin sections was aided by viewing large areas of the luminal sinusoidal cell surface in secondary electron (SEI) and backscattered electron imaging (BEI) modes of the scanning electron microscope.

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.

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).

Backscattered Electron Imaging of Colloidal Gold Markers For Leukocyte Surface Antigens

1985 ◽  
Vol 43 ◽  
pp. 534-537
Author(s):  
Etienne de Harven ◽  
Davide Soligo
Keyword(s):  
Colloidal Gold ◽  
Bacteriophage T4 ◽  
Small Cell ◽  
Surface Structures ◽  
Coated Pits ◽  
Backscattered Electron Imaging ◽  
Cell Surface Structures ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
20 Nm

Markers for immuno-scanning electron microscopy had been, so far, selected for easy identification based on their distinctive shape (for example, haemocyanin, bacteriophage T4). These markers were always viewed in the secondary electron imaging (SEI) mode. Their size was not posing much of a problem of resolution for commercially available SEM, but was likely to prevent good labeling efficiency, due to steric hindrance phenomena. Higher labeling efficiency necessitates the use of markers of smaller size which, unfortunately, can rarely be unambiguously recognized in topographical SE images.To correlate antigenic distribution with small cell surface structures like microvilli or coated pits, markers of smaller size (i.e., in the 20 nm range) must be used and clearly identified on the surface of well preserved cells. With the availability of colloidal gold particles complexed with various ligands this became possible, particularly if the labeled cells are viewed in the backscattered imaging (BEI) mode of the SEM, therefore basing the identification of the marker on its atomic number contrast rather than on its topographical contrast.

Sinusoidal Cells in Bone Marrow Take Up BSA-Au Conjugates in the Presence of an Artificial Blood Substitute

1985 ◽  
Vol 43 ◽  
pp. 700-701
Author(s):  
J.S. Geoffroy ◽  
R.P. Becker
Keyword(s):  
Bone Marrow ◽  
Los Angeles ◽  
Iliac Artery ◽  
Blood Substitute ◽  
Sprague Dawley ◽  
Coated Pits ◽  
Sinusoidal Cells ◽  
Artificial Blood ◽  
Left Common Iliac Artery

The pattern of BSA-Au uptake in vivo by endothelial cells of the venous sinuses (sinusoidal cells) of rat bone marrow has been described previously. BSA-Au conjugates are taken up exclusively in coated pits and vesicles, enter and pass through an “endosomal” compartment comprised of smooth-membraned tubules and vacuoles and cup-like bodies, and subsequently reside in multivesicular and dense bodies. The process is very rapid, with BSA-Au reaching secondary lysosmes one minute after presentation. (Figure 1)In further investigations of this process an isolated limb perfusion method using an artificial blood substitute, Oxypherol-ET (O-ET; Alpha Therapeutics, Los Angeles, CA) was developed. Under nembutal anesthesia, male Sprague-Dawley rats were laparotomized. The left common iliac artery and vein were ligated and the right iliac artery was cannulated via the aorta with a small vein catheter. Pump tubing, preprimed with oxygenated 0-ET at 37°C, was connected to the cannula.

High-sensitivity detection of gold labels by high-angle dark-field STEM imaging

1990 ◽  
Vol 48 (3) ◽  
pp. 892-893 ◽  
Author(s):  
Max T. Otten
Keyword(s):  
High Sensitivity ◽  
Dark Field ◽  
Bright Field ◽  
Backscattered Electron Imaging ◽  
Backscattered Electrons ◽  
Average Atomic Number ◽  
Highly Sensitive ◽  
Backscattered Electron ◽  
Electron Imaging ◽  
High Sensitivity Detection

Labelling of antibodies with small gold probes is a highly sensitive technique for detecting specific molecules in biological tissue. Larger gold probes are usually well visible in TEM or STEM Bright-Field images of unstained specimens. In stained specimens, however, the contrast of the stain is frequently the same as that of the gold labels, making it virtually impossible to identify the labels, especially when smaller gold labels are used to increase the sensitivity of the immunolabelling technique. TEM or STEM Dark-Field images fare no better (Figs. 1a and 2a), again because of the absence of a clear contrast difference between gold labels and stain.Potentially much more useful is backscattered-electron imaging, since this will show differences in average atomic number which are sufficiently large between the metallic gold and the stains normally used. However, for the thin specimens and at high accelerating voltages of the STEM, the yield of backscattered electrons is very small, resulting in a very weak signal. Consequently, the backscattered-electron signal is often too noisy for detecting small labels, even for large spot sizes.

Enzyme histochemical application and backscattered electron imaging to visualize the distribution of lymphatic capillaries

1990 ◽  
Vol 48 (3) ◽  
pp. 880-881
Author(s):  
Seiji Kato
Keyword(s):  
Staining Method ◽  
Double Staining ◽  
Reaction Products ◽  
Blood Capillaries ◽  
Backscattered Electron Imaging ◽  
Histochemical Observation ◽  
Backscattered Electron ◽  
Double Staining Method ◽  
Electron Imaging ◽  
Cryostat Sections

Previously, the author repeatedly confirmed the higher 5’-nucleotidase (5’-Nase) and lower alkaline phoaphatase (ALPase) activities in the wall of lymphatic capillaries reacted with the lead-based method relative to those of blood capillaries. The ALPase, on the other hand, is markedly higher in blood capillaries than in lymphatics. On the basis of these enzyme characteristics, the author has developed a 5’-Nase— ALPase double staining method to differentiate small lymphatics from blood capillaries at the level of the light microcsopy. Furthermore, we applied it to histochemical observation of the lead-containing reaction products of 5’-Nase in lymphatics on the same or adjacent cryostat sections using backscattered electron imaging (BEI) in scanning electron microscope (SEM). This paper presents a new applicability of 5’-Nase histochemistry by BEI-SEM to demonstrate the distribution of lymphatic capillaries in tissue blocks.

scholarly journals Osteoconductive Properties of a Volume-Stable Collagen Matrix in Rat Calvaria Defects: A Pilot Study

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 732
Author(s):  
Karol Alí Apaza Alccayhuaman ◽  
Stefan Tangl ◽  
Stéphane Blouin ◽  
Markus A. Hartmann ◽  
Patrick Heimel ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bone Formation ◽  
Collagen Matrix ◽  
Microcomputed Tomography ◽  
Soft Tissue Augmentation ◽  
Backscattered Electron Imaging ◽  
Quantitative Backscattered Electron Imaging ◽  
Rat Calvaria ◽  
Backscattered Electron ◽  
Electron Imaging

Volume-stable collagen matrices (VSCM) are conductive for the connective tissue upon soft tissue augmentation. Considering that collagen has osteoconductive properties, we have investigated the possibility that the VSCM also consolidates with the newly formed bone. To this end, we covered nine rat calvaria circular defects with a VSCM. After four weeks, histology, histomorphometry, quantitative backscattered electron imaging, and microcomputed tomography were performed. We report that the overall pattern of mineralization inside the VSCM was heterogeneous. Histology revealed, apart from the characteristic woven bone formation, areas of round-shaped hypertrophic chondrocyte-like cells surrounded by a mineralized extracellular matrix. Quantitative backscattered electron imaging confirmed the heterogenous mineralization occurring within the VSCM. Histomorphometry found new bone to be 0.7 mm2 (0.01 min; 2.4 max), similar to the chondrogenic mineralized extracellular matrix with 0.7 mm2 (0.0 min; 4.2 max). Microcomputed tomography showed the overall mineralized tissue in the defect to be 1.6 mm3 (min 0.0; max 13.3). These findings suggest that in a rat cranial defect, VSCM has a limited and heterogeneous capacity to support intramembranous bone formation but may allow the formation of bone via the endochondral route.

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