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PubMedDate not supplied

Detection of gold probes with video-enhanced contrast microscopy: nanovid microscopy.

This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20-40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor-mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell-surface glycoproteins on PTK2-cells. In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.

Biological Transport↗
PubMed1992

Mammotropes and somatotropes in the adenohypophysis of androgenized female mice: morphological and immunohistochemical studies by light microscopy correlated with routine electron microscopy.

Female mice were divided into androgenized (AF) and control (CF) groups. The AF mice were injected subcutaneously with testosterone propionate (Tp) and the CF mice with sesame oil at 5 days of age. Mammotropes (PRL cells) and somatotropes (GH cells) in the adenohypophyses of these mice when they became adults were studied with immunohistochemistry and morphometry by light microscopy correlated with routine electron microscopy. In CF mice, almost all of the PRL-immunoreactive cells (about 43% of all parenchymal cells) were type I (classical) PRL cells, and almost all of the GH-immunoreactive cells (about 30% of all parenchymal cells) were type I (classical) GH cells. Type II PRL cells accounted for about 0.4% of parenchymal cells, and type II GH cells were about 2.5% of all parenchymal cells. In AF mice, the percentages of PRL and GH cells were not significantly different from those of CF mice. Mammosomatotropes (Ms cells) in both groups were less than 1% of all parenchymal cells. Numbers of all parenchymal, PRL and GH cells, however, were increased significantly in AF mice when compared to those in CF, because the adenohypophysis was increased in volume in AF mice. Type I PRL cells were larger in AF than in CF. The ultrastructure suggested that type I PRL cells may show increased PRL synthesis and secretion in AF mice. Furthermore, AF mice, in which the hypothalamus is masculinized by the neonatal treatment with Tp, retained feminine characteristics in the population and size of PRL cells and GH cells in the adenohypophysis.

Androgens↗
PubMed2004

Magnetic resonance microscopy versus light microscopy in human embryology teaching.

A study was carried out on the application of magnetic resonance microscopy (MRM) in teaching prenatal human development. Human embryos measuring 8 mm, 15 mm, 18.5 mm, and 22 mm were fixed in a 4% paraformaldehyde solution and sections obtained with magnetic resonance imaging (MRI) were compared to those prepared for light microscopy (LM), using the same embryos. The MRM and LM slices were of a similar quality. In the MRM sections, embryonic organs and systems were clearly visible, particularly the peripheral and central nervous systems, and the cardiovascular and digestive systems. The digitalization and clarity of the MRM images make them an ideal teaching aid that is suitable for students during the first years of a health-science degree, particularly medicine. As well as providing students with their first experience of MRM, these images allow students to access, at any time, all embryos used, to assess changes in the positions of different organs throughout their stages of development, and to acquire spatial vision, an absolute requirement in the study of human anatomy. We recommend that this technique be incorporated into the wealth of standard embryonic teaching methods already in use.

Embryo, Mammalian↗
PubMed1992

High-voltage electron microscopy and conventional transmission electron microscopy of the interface zone between bone and endosteal dental implants.

The interface between mandibular bone and endosteal dental implants was examined with an in vivo dog model. Undecalcified mandibular implant samples were observed with both conventional transmission electron microscopy and high-voltage transmission electron microscopy (HVEM). Results demonstrated the variable nature of the interfacial support tissues. Mineralized bone was often found within 50 nm of the implant surface, separated from that surface only by an electron dense deposit. Osteocytes were observed close to the interface encased within lacunae extending numerous cellular processes through canaliculi. An osteoblast was also observed directly at the interface within a developing lacuna. Other interfacial areas exhibited a finely fibrillar and more electron lucent morphology. Furthermore, other areas were shown to be composed of wider zones of extracellular products containing collagen fibrils, ground substance, and calcified inclusions. Because bone is an actively growing and remodeling tissue, these different morphological zones around the entire area of the implants would appear to confirm the dynamic tissue response to endosteal dental implants. Further, HVEM stereology was shown to be an exciting research tool to investigate this tissue response.

Animals↗
PubMed1998

Investigation of cholesterol, bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy.

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallastones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue-cholesterol, red, orange-bilirubin, yellow, green-protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultrathin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.

Bilirubin↗
PubMed1997

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy.

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 micron Araldite-embedded histologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 micron Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and "expansion chambers" may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.

Aqueous Humor↗
PubMed1979

Glomerulonephritis induced by high doses of ovalbumin. Studies by electron microscopy, immunofluorescence and immuno-electron microscopy.

Experimental glomerulonephritis was produced in 16 rabbits by intravenous injections of ovalbumin in high doses (0.1 g/day during the first week, 0.2 g x 6/day during the second). The animals were killed on day 14. At that time all animals had 2--4+ proteinuria and a serum C3 level reduced to about 50% of the control level; 11 animals had a significantly raised blood urea level. In all rabbits the antigen had induced severe proliferative glomerulonephritis. Electron microscopy showed that many of the cells accounting for the hypercellularity were monocytes. Surprisingly, electron dense deposits were few and small, mainly on the subendothelial and subepithelial aspects of the glomerular basement membrane. In all the animals ultrastructural immunoperoxidase technique revealed deposits containing ovalbumin, rabbit IgG and C3. With immunofluorescence sparse deposits were occasionally seen. It is concluded that a severe experimental glomerulonephritis can be produced in a state of antigen excess, with the deposition of immune complexes being minimal. Immuno-electron microscopy is essential, however, in detecting even the smallest animals of deposited immune reactants.

Animals↗
PubMed1983

Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. I. X-ray diffraction monitoring during preparation for electron microscopy of muscle fibres fixed in rigor, in ATP and in AMPPNP.

Synchrotron radiation was used for low-angle X-ray diffraction to monitor structural changes produced in insect flight muscle during fixation, dehydration and embedding for electron microscopy of thin sections. Fibre bundles were fixed by cold glutaraldehyde in one of three states, namely rigor, ATP or AMPPNP, followed by additional cross-linking treatment. No heavy metals were used before embedding. During fixation-embedding, all specimens lost the continuous actin layer lines of spacing 11-5 nm, shrank 18-21% in lattice spacing, shrank 0.5-2.5% in axial spacings and showed equatorial intensity changes which were similar for all three states, while the well-sampled inner layer lines (39-13 nm) were preserved with different fidelity in each state, highest for rigor and lowest for ATP. In different AMPPNP bundles, these layer lines indicated different degrees of unexplained shift (from slight to total) towards the structure of muscle fixed in ATP. Fixation in ATP caused obvious gain of intensity on 39, 19 and 13 nm layer lines, which can be interpreted as trapping of myosin crossbridge attachments to actin; this artifact was unchanged by seven variations in fixation conditions. Fixation in rigor gave no indication of crossbridge detachment nor of the presence or alteration of any significant population of non-bridging myosin heads. X-ray monitoring allowed selection of best-preserved samples for subsequent electron microscopy. The rapid pattern-recording possible with synchrotron X-ray intensity allowed us to complete and compare experiments with many fibre bundles from a single glycerinated Lethocerus muscle.

Adenosine Triphosphate↗
PubMed1993

Bone ingrowth into hydroxyapatite coating: a light microscopy and laser scanning microscopy study.

The authors present a description of the presence of organic material inside the hydroxyapatite (HA) coating of Calcitek implants inserted in rabbit tibia. Light microscopy showed that in all specimens a granular basophilic material which was not mineralized was present inside the thickness of the coatings. In two specimens out of 40, a bone-like substance was observed in some areas of the coating, at a distance from the interface. Laser scanning microscopy showed in all specimens an autofluorescence of osteocytes, osteoblasts, of the interface, and inside the coating. The presence of bone could either show an ability of the coating to serve as a scaffold for the newly forming osseous tissue or be a sign of an initial degradation of the HA.

Animals↗
PubMed1984

A simplified microwell pseudoreplica for the detection of viruses by electron microscopy and immunoelectron microscopy.

Simplified procedures for immunoelectron microscopy (IEM) and electron microscopy (EM) are described. The procedures employ the principle of agar filtration and pseudoreplication. The modification consisted of the use of microwells for storage of gels with or without antiserum (for IEM or EM, respectively) and an array of containers in which pseudoreplication and negative staining were performed. The containers were prepared from 5 ml syringes from which the needle holding parts were cut. This device enabled simultaneous and rapid handling of specimens. With Sindbis virus as a model, our microwell pseudoreplica IEM (MW-PR-IEM) was compared to six other IEM techniques and was found to be the most rapid and sensitive technique. With the MW-PR-IEM technique, the specific minimal detection limit (detection of clumps) was 1.5 x 10(7) virus particles per ml, and the non-specific detection limit (detection of single virions) was 1.8 x 10(6) virus particles per ml.

Antibodies, Viral↗
PubMed1984

Structure and composition of microcalcifications in benign and malignant lesions of the breast: study by light microscopy, transmission and scanning electron microscopy, microprobe analysis, and X-ray diffraction.

Microcalcifications previously located by radiography were extracted from 25 fresh specimens obtained from patients who had undergone tumorectomy or systematized mammary exeresis. Two principal types of microcalcifications were distinguished: Type I microcalcifications were amber in color and generally crystalline on scanning electron microscopy, with only one calcium peak on microprobe analysis; x-ray diffraction revealed that weddellite was involved. Type II microcalcifications were whitish, nonbirefringent under polarized light, and generally ovoid or fusiform, with two peaks, one calcium and the other phosphorus, on microprobe analysis; these microcalcifications were composed of calcium phosphate, the most characteristic form of which is hydroxyapatite, in the form of needles arranged in rosettes on transmission electron microscopy. Type I microcalcifications were observed in four of eight benign breast lesions, in two of three in situ lobular carcinomas, and in no intraductal adenocarcinomas or infiltrating carcinomas. Type II microcalcifications were present in all infiltrating carcinomas and intraductal adenocarcinomas; they were also found in benign lesions (four of eight) and even associated with type I microcalcifications in one in situ lobular carcinoma. There are, therefore, no "benign" or "malignant" microcalcifications; however, the presence of weddellite is a strong indication that a lesion is benign or, at most, an in situ lobular carcinoma.

Adenocarcinoma↗
PubMed1989

Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy.

The results and anecdotes presented here are intended only as a general guide to other would-be immunocytochemists, because other proteins will undoubtedly respond at least somewhat differently than does HMG-CoA reductase. Nevertheless, based on these experiences, we offer the following suggestions: 1. Antiserum of high specificity should be raised and affinity-purified. Using this antiserum, immunofluorescence microscopy should be attempted before resorting to electron microscopic localization. In the absence of immunolocalization at the light-microscope level, it may be a waste of time to pursue the problem to higher levels of resolution. 2. Cells should be prefixed in 1% formaldehyde-1% glutaraldehyde. Direct fixation of the growing culture and use of phosphate buffer are recommended. The prefixed sample can then be divided into two or three aliquots. One aliquot should receive no postfixation (for optimal immunoreactivity), while the others can be postfixed in osmium-potassium ferricyanide (for possible immunolocalization) or permanganate (for ultrastructural analysis). Because of its ease of use, Spurr's resin should be tried initially. If immunocytochemistry is successful, no further preparations are necessary. If unsuccessful, LR White resin is recommended, but the sample must be treated to remove the cell wall. Electron microscopy and immunocytochemistry offer views into the molecular arrangement of individual cells, a view not easily obtained by other means. It is satisfying and often enlightening to be able to see the extremes as well as the average. In studies of the organization of karmellae, for example, ultrastructural analysis easily revealed the asymmetric segregation pattern, while immunoblots and cell fractionation could not even demonstrate the existence of this membrane organization. The richness of the information available to those who can avert reductionist tendencies, even for a short time, is remarkable.

Histocytochemistry↗
PubMed1980

Electron microscopy: a method for the diagnosis of inherited metabolic storage diseases. Electron microscopy in diagnosis.

In the diagnosis of inherited metabolic diseases, electron microscopy has become an important method complementary to clinical, histological and biochemical assays. The characteristic ultrastructure of stored material as well as the site of accumulation in the cell are shown in a number of metabolic disorders. The most prominent advantages of electron microscopical techniques as compared to alternative techniques are discussed. One of the advantages is the fact that ultrastructural investigation requires only tissue samples of very small size, and another that its results may be obtained within two days. Moreover, transmission electron microscopy permits new and promising analytical methods such as quantitative estimation of organellar changes (morphometry) and energy dispersive X-ray elemental analysis (EDX).

Fabry Disease↗
PubMed1999

Modern microscopy in biofilm research: confocal microscopy and other approaches.

Microscopy is the only technique whereby bacterial biofilms can be studied at the single-cell level in situ. Our understanding of biofilm structure, physiology and control hinges on the application of confocal scanning laser microscopy and other advanced microscopic techniques. Gene expression in four dimensions (x,y,z,t), interspecies interactions, and the role of exopolymer are being defined.

Biofilms↗
PubMed2003

Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM).

In order to evaluate the potential use of environmental scanning electron microscopy (ESEM) in biology, structural changes of the jejunal villi of rats were studied after periods of fasting and refeeding, using a conventional scanning electron microscope (CSEM) and ESEM. While observation using the CSEM, involves chemical fixation, drying and coating, observation of fresh, unprepared materials can be directly realized with the ESEM. Environmental microscopy provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. Direct observation of biological samples in their native state is therefore possible with an ESEM. After fasting, the jejunal mucosa is dramatically reduced in size, splits and holes appearing at the tip of the villi. These changes were observed whatever the type of technique used. Artifacts due to the sample preparation for CSEM observation (drying, coating) can therefore be excluded. However, CSEM and ESEM must be used jointly. While, CSEM must be preferred for surface analysis involving high magnifications, ESEM observation, on the other hand, can prove valuable for determining the living aspect of the samples.

Animals↗
PubMed1998

Atomic force microscopy and other scanning probe microscopies.

The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy.

Annexin A5↗
PubMed2001

Specific surface area of snow samples determined by CH4 adsorption at 77 K and estimated by optical microscopy and scanning electron microscopy.

Snow is a divided medium that can adsorb atmospheric trace gases. Evaluating the impact of the snow cover on atmospheric chemistry therefore requires the knowledge of the specific surface area (SSA) of snow. This paper compares the results of three methods used to measure or estimate the SSA of four snow samples: CH4 adsorption at 77 K, optical microscopy (OM), and scanning electron microscopy (SEM, used only on two samples). Within error bars, CH4 adsorption and OM yield similar results on three of the four snow samples. Values for the 4th sample are within a factor of 2. For both samples where CH4 adsorption, OM, and SEM are used, all three methods yield similar results, but CH4 adsorption always has a better accuracy and a much better precision. Thus, despite its ease of use, estimates from OM images are often not accurate enough to monitor the evolution of snow SSA. The main sources of error in the OM method are the difficulty to determine snow crystal thicknesses and to take into account the topography of the snow crystal surface. The combination of CH4 adsorption and OM or SEM can provide useful information on the evolution of both the SSA and the shape of snow crystals. This will be useful to evaluate the respective contributions of adsorption/desorption and sublimation/condensation processes to the impact of the snow cover on atmospheric chemistry.

Adsorption↗
PubMed2001

Effect of bipolar radiofrequency energy on human articular cartilage. Comparison of confocal laser microscopy and light microscopy.

PURPOSE: To evaluate chondrocyte viability using confocal laser microscopy (CLM) following exposure to bipolar radiofrequency energy (bRFE) and to contrast CLM with standard light microscopy (LM) techniques. TYPE OF STUDY: In vitro analysis using chondromalacic human cartilage. METHODS: Twelve fresh chondral specimens were treated with the ArthroCare 2000 bRFE system (ArthroCare, Sunnyvale, CA) coupled with 1 of 2 types of probes and at 3 energy delivery settings (S2, S4, S6). A sham-operated group was treated with no energy delivered. Specimens were analyzed for chondrocyte viability and chondral morphology with CLM using fluorescent vital cell staining and with LM using H&E and safranin-O staining. RESULTS: LM with H&E staining showed smoothing of fine fronds of fibrillated cartilage; thickened fronds were minimally modified. Chondrocyte nuclei were present and not morphologically different than nuclei within sham-operated and adjacent untreated regions. LM with safranin-O staining showed a clear demarcation between treated and untreated regions. CLM, however, showed chondrocyte death: the depth and width of chondrocyte death increased with increasing bRFE settings. CONCLUSIONS: CLM showed that bRFE delivered through the probes investigated created significant chondrocyte death. These changes were not apparent using LM techniques.

Aged↗