scholarly journals Light microscopic morphometric analysis of peroxisomes by automatic image analysis: advantages of immunostaining over the alkaline DAB method.

1992 ◽  
Vol 40 (1) ◽  
pp. 115-121 ◽  
Author(s):  
K Beier
Keyword(s):  
Image Analysis ◽  
Light Microscopy ◽  
Morphometric Analysis ◽  
Protein A ◽  
Volume Density ◽  
Section Thickness ◽  
Automatic Image Analysis ◽  
Electron Microscopic ◽  
Lr White ◽  
Electron Microscopic Morphometry

The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.

scholarly journals A Simple Method to Estimate the Number of Autophagic Elements by Electron Microscopic Morphometry in Real Cellular Dimensions

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Attila L. Kovács
Keyword(s):  
Surface Density ◽  
Average Diameter ◽  
Volume Density ◽  
Cell Type ◽  
Electron Microscopic ◽  
Simple Method ◽  
Density Data ◽  
Pancreatic Cells ◽  
Electron Microscopic Morphometry

Autophagic elements typically appear as spherical bodies. During their life they undergo a series of changes (e.g., fusion, degradation of content, and swelling) which influence their size in a way that may be characteristic for cell type, stage of maturation, or various experimentally manipulated parameters. A simple and time efficient method is suggested here to use exactly calculated specific surface values and estimate average diameter and number of autophagic elements in real cellular dimensions. The method is based on the easiest morphometric determination of relative surface (surface density) and volume (volume density) data by electron microscopy. A series of data from real experimental samples of liver and exocrine pancreatic cells are offered to illustrate the potential of these measurements and calculations.

scholarly journals Immunocytochemical localization of ornithine transcarbamylase in rat intestinal mucosa. Light and electron microscopic study.

1988 ◽  
Vol 36 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Y Hamano ◽  
H Kodama ◽  
M Yanagisawa ◽  
Y Haraguchi ◽  
M Mori ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Light Microscopy ◽  
Intestinal Mucosa ◽  
Intestinal Epithelial Cells ◽  
Protein A ◽  
Intestinal Epithelial ◽  
Electron Microscopic ◽  
Small Intestinal

We investigated light and electron microscopic localization of ornithine transcarbamylase (OTC) in rat intestinal mucosa. In the immunoblotting assay of OTC-related protein, a single protein band with a molecular weight of about 36,500 is observed in extracts of liver and small intestinal mucosa but is not observed in those of stomach and large intestine. For light microscopy, tissue slices of the digestive system were embedded in Epon and stained by using anti-bovine OTC rabbit IgG and the immunoenzyme technique. For electron microscopy, slices of these and the liver tissues were embedded in Lowicryl K4M and stained by the protein A-gold technique. By light microscopy, the absorptive epithelial cells of duodenum, jejunum, and ileum stained positively for OTC, but stomach, large intestine, rectum, and propria mucosa of small intestine were not stained. Electron microscopy showed that gold particles representing the antigenic sites for OTC were confined to the mitochondrial matrix of hepatocytes and small intestinal epithelial cells. However, the enzyme was detected in mitochondria of neither liver endothelial cells, submucosal cells of small intestine, nor large intestinal epithelial cells. Labeling density of mitochondria in the absorptive epithelial cells of duodenum, jejunum, and ileum was about half of that in liver cells.

Ultrastructural changes in rat hepatocytes after partial hepatectomy, and comparison with biochemical results

1981 ◽  
Vol 50 (1) ◽  
pp. 433-448
Author(s):  
A.B. Murray ◽  
W. Strecker ◽  
S. Silz
Keyword(s):  
Partial Hepatectomy ◽  
Specific Effect ◽  
Rat Hepatocytes ◽  
Volume Density ◽  
Ultrastructural Changes ◽  
Sham Operation ◽  
Electron Microscopic ◽  
Electron Microscopic Morphometry ◽  
Mitochondrial Volume ◽  
Massive Accumulation

Ultrastructural changes in rat hepatocytes in the first 24 h following partial hepatectomy (p.h.), i.e. in the premitotic phase of liver regeneration, were studied using electron microscopic morphometry. Livers were investigated 1/2, 1, 4, 8, 12, 20 and 24 h after p.h. and 1/2, 8, 20 and 24 h after a sham operation. Two effects appear to be associated specifically with the regeneration process: (1) an increase in the volume density of lysosomes to a peak between 4 and 8 h after p.h.; and (2) a doubling in the number of mitochondria per cell by 24 h without any associated increase in the total mitochondrial volume. Two further changes were observed only after p.h.: (1) a massive accumulation of lipid in the form of lipid droplets by 24 h; and (2) the appearance of ‘protein droplets’ (very large lysosomal-like structures) at various stages. Both these changes appear to be secondary effects associated with the stimulation for, but not necessary to, cell division. The loss of glycogen observed immediately after both p.h. and a sham operation is a non-specific effect probably resulting from operation-induced stress. The results are discussed with reference to the changes observed in the biochemical composition of blood plasma. Glucagon appears to play an important role in the stimulation of some of the ultrastructural changes observed.

Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification

2017 ◽  
Vol 122 (4) ◽  
pp. 1019-1030 ◽  
Author(s):  
Julia Schipke ◽  
Christina Brandenberger ◽  
Alexandra Rajces ◽  
Martin Manninger ◽  
Alessio Alogna ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Light Microscopy ◽  
Cardiac Fibrosis ◽  
Early Stage ◽  
Method Comparison ◽  
Automated Image Analysis ◽  
Section Thickness ◽  
Quantification Method ◽  
Em Stereology

Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson’s trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining. NEW & NOTEWORTHY Direct comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes.

A New System For Optomanual Semiautomatic Quantitative Image Analysis

1977 ◽  
Vol 35 ◽  
pp. 210-211
Author(s):  
H.P. Rohr
Keyword(s):  
Image Analysis ◽  
Volume Density ◽  
List Type ◽  
Distribution Analysis ◽  
Type Number ◽  
Point Counting ◽  
Human Eye ◽  
Quantitative Image ◽  
Point Density ◽  
Electronic Counting

Today, in image analysis the broadest possible rationalization and economization have become desirable. Basically, there are two approaches for image analysis: The image analysis through the so-called scanning methods which are usually performed without the human eye and the systems of optical semiautomatic analysis completely relying on the human eye.The new MOP AM 01 opto-manual system (fig.) represents one of the very promising approaches in this field. The instrument consists of an electronic counting and storing unit, which incorporates a microprocessor and a keyboard for choice of measuring parameters, well designed for easy use.Using the MOP AM 01 there are three possibilities of image analysis:the manual point counting,the opto-manual point counting andthe measurement of absolute areas and/or length (size distribution analysis included).To determine a point density for the calculation of the corresponding volume density the intercepts lying within the structure are scanned with the light pen.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

Correlative video enhanced differential interference contrast light microscopy (VDIC)-LM), low-voltage high-resolution SEM (LV-HR-SEM), and high-voltage electron microscopy (HVEM) in the observation of gold-labelled surface antigens and the subjacent cytoskeletal matrix

1989 ◽  
Vol 47 ◽  
pp. 904-905
Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
Marek Malecki
Keyword(s):  
High Resolution ◽  
Light Microscopy ◽  
Low Voltage ◽  
Microscopic Analysis ◽  
Cell Labelling ◽  
Video Technology ◽  
Electron Microscopic ◽  
Image Capture ◽  
High Voltage Electron Microscopy ◽  
Fluorescence Studies

The development of video-enhanced light microscopy (LM) as well as associated image processing and analysis have significantly broadened the scope of investigations which can be undertaken using (LM). Interference/polarization based microscopies can provide high resolution and higher levels of “detectability” especially in unstained living systems. Confocal light microscopy also holds the promise of further improvements in resolution, fluorescence studies, and 3 dimensional reconstruction. Video technology now provides, among other things, a means to detect differences in contrast difficult to detect with the human eye; furthermore, computerized image capture, processing, and analysis can be used to enhance features of interest, average images, subtract background, and provide a quantitative basis to studies of cells, cell features, cell labelling, and so forth. Improvements in video technology, image capture, and cost-effective computer image analysis/processing have contributed to the utility and potential of the various interference and confocal microscopic instrumentation.Electron microscopic technology has made advances as well. Microprocessor control and improved design have contributed to high resolution SEMs which have imaging capability at the molecular level and can operate at a range of accelerating voltages starting at 1KV. Improvements have also been seen in the HVEM and IVEM transmission instruments. As a whole, these advances in LM and EM microscopic technology provide the biologist with an array of information on structure, composition, and function which can be obtained from a single specimen. Corrrelative light microscopic analysis permits examination of living specimens and is critical where the “history” of a cell, cellular components, or labels needs to be known up to the time of chemical or physical fixation. Features such as cytoskeletal elements or gold label as small as 0.01 μm, well below the 0.2 μm limits of LM resolution, can be “detected” and their movement followed by VDIC-LM. Appropriate identification and preparation can then lead to the examination of surface detail and surface label with stereo LV-HR-SEM. Increasing the KV in the HR-SEM while viewing uncoated or thinly coated specimens can provide information from beneath the surface as well as increasing Z contrast so that positive identification of surface and subsurface colloidal gold or other heavy metal labelled/stained material is possible. Further examination of the same cells using stereo HVEM or IVEM provides information on internal ultrastructure and on the relationship of labelled material to cytoskeletal or organellar distribution, A wide variety of investigations can benefit from this correlative approach and a number of instrumentational configurations and preparative pathways can be tailored for the particular study. For a surprisingly small investment in time and technique, it is often possible to clear ambiguities or questions that arise when a finding is presented in the context of only one modality.

Radial mass analysis of unstained STEM images of molluscan hemocyanins

1990 ◽  
Vol 48 (3) ◽  
pp. 810-811
Author(s):  
M.G. Hamilton ◽  
T.T. Herskovits ◽  
J.S. Wall
Keyword(s):  
Image Analysis ◽  
Hollow Cylinder ◽  
Electron Micrographs ◽  
Side View ◽  
Mass Analysis ◽  
Mass Measurements ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Transmission Electron Microscopic ◽  
Microscopic Studies

The hemocyanins of molluscs are aggregates of a cylindrical decameric subparticle that assembles into di-, tri-, tetra-, penta-, and larger multi-decameric particles with masses that are multiples of the 4.4 Md decamer. Electron micrographs of these hemocyanins typically show the particles with two profiles: circular representing the cylinder viewed from the end and rectangular representing the side-view of the hollow cylinder.The model proposed by Mellema and Klug from image analysis of a didecameric hemocyanin with the two decamers facing one another with collar (closed) ends outward fits the appearance of side-views of the negatively-stained cylinders. These authors also suggested that there might be caps at the ends. In one of a series of transmission electron microscopic studies of molluscan hemocyanins, Siezen and Van Bruggen supported the Mellema-Klug model, but stated that they had never observed a cap component. With STEM we have tested the end cap hypothesis by direct mass measurements across the end-views of unstained particles.

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