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PubMed2003

The outer integument and funicular outgrowth complex in the ovule of Magnolia grandiflora (Magnoliaceae).

The development of the outer integument and funicular outgrowth in the ovule of Magnolia grandiflora was examined by microtomy and scanning electron microscopy to reveal the morphology and evolution of the outer integument, a novel angiosperm structure. Early in development the outer integument is semiannular, decurrent to the lateral sides of the funiculus, and extends downwards beyond the funicular outgrowth that forms in the gap of the outer integument, and is transverse to the funiculus. The outer integument then overgrows the funicular outgrowth perpendicularly to the funiculus to form a micropyle together. The hood-shaped outer integument and the funicular outgrowth compose an envelope complex, and the interpretation of a single cupular outer integument is not supported. This envelope complex may differ from the cupular outer integument of other angiosperms, e.g., Nymphaeaceae, suggesting independent origin of apparently cupular outer integuments and hood-shaped outer integuments. Anatropous curving is due mainly to differential growth of the chalaza. The bistomic micropyle of Magnoliaceae seems to represent a derived character state, compared to an endostomic micropyle.

Magnolia↗
PubMed1992

Freeze-substitution studies of bacteria.

Typically, models of bacterial structure combine biochemical data obtained from bulk analyses of cell populations with electron microscopic observation of individual cells. Recent development of a battery of cryotechniques specific for biological electron microscopy have begun to supercede routine procedures such as conventional thin sectioning. One of these cryotechniques, freeze-substitution, combines the advantages of ultrarapid freezing with standard microtomy methods. This technique is particularly well suited to the examination of bacterial structure and has yielded additional ultrastructural information consistent with biochemical data but often challenging models of cell structure obtained from conventional microscopical methods. In addition to retaining more accurately the spatial distribution of cell components, freeze-substitution has been successfully combined with immunochemical labelling techniques and has enabled identification and localization of specific molecules both within the cell and on the cell surface. In this review, I describe current ideas on bacterial ultrastructure, modified in accordance with new data obtained from recent freeze-substitution studies.

Bacteria↗
PubMed2004

Cross-section analysis of organic light-emitting diodes.

The 'lift-out' technique using a focused ion beam microscope was applied to prepare cross-sectional specimens of organic light-emitting diodes for use in transmission electron microscopy. The focused ion beam equally thins the organic/inorganic hybrid devices despite the difference in material hardness of the compounds. This allowed to overcome preparation difficulties of conventional techniques such as ion thinning or ultra-microtomy. Two different samples were prepared and studied by both conventional transmission electron microscopy and analytical electron microscopy to display some of the investigation possibilities which become available with this sample preparation method.

Aluminum↗
PubMedDate not supplied

Three-dimensional imaging of biological complexity.

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.

Animals↗
PubMed2006

Role of stapes surgery in improving hearing loss caused by otosclerosis.

The aim of our study was to evaluate the functional results of stapes surgery and to compare the effectiveness of small fenestra stapedotomy with that of total stapedectomy in improving hearing in patients affected by otosclerosis. Three hundred and fifty-seven consecutive ears, in 265 patients affected by otosclerosis, underwent surgery. All cases underwent either primary small fenestra stapedotomy (group A, 196/357, 54.91 per cent) or stapedectomy (group B, 161/357, 45.09 per cent). After surgery, 256/357 (71.71 per cent) cases showed a 0-20 dB gap. There were no significant differences in hearing results between the two groups at either early or late post-operative assessment. The mean post-operative pure tone average and air-bone gap results were slightly greater for group B than for group A, at both early and late post-operative assessments, but these differences were not statistically significant. Therefore, in group A, the mean pure tone average at 4 kHz significantly improved, from 56.60 to 47.66 dB at early post-operative assessment and to 52.98 dB at late post-operative assessment. Our study suggests that the technique of microtomy of the oval window is able to improve hearing results especially at high frequencies.

Adult↗
PubMed2007

Assessing the macroporous structure of monolithic columns by transmission electron microscopy.

A set of monolithic stationary phases representing a broad span of monomers and porogens have been characterized directly in their capillary chromatographic format by computational assessment of their pore structure from transmission electron micrographs obtained after in situ embedment of the monoliths in contrast resin, followed by dissolution of the fused-silica tubing, further encasement of the resin-embedded monolith, and microtomy. This technique has been compared to mercury intrusion, a more conventional technique for macroporosity estimation. Supplementing the embedding resin by lead methacrylate gave a negative staining, and the resulting micrographs showed a good contrast between the polymeric monoliths and the embedding resin that allowed studies on the pore formation and polymer development. The technique was also applied to a commercial monolithic silica column.

Journal Article↗
PubMed2001

Quantitative analysis of intravitreal injections in the rat.

Intravitreal injections are currently used in the rat to introduce a therapeutic factor in the eye, especially for experimental treatments of retinal degenerations. The injected volume and its location can influence the quantification of results. We have investigated the quantitative effect of a single intravitreal injection in rats at different ages and for different volumes. Albinos rats aged three weeks or two months received intravitreal injections of 1, 3, 5 or 10 microl China ink. Animals were sacrificed immediately after injection, eyes were enucleated, fixated, embedded in paraffin and microtomy was performed in a sagittal plane. Regularly spaced sections were analyzed to reconstruct the vitreous and injected dye volumes. The measured vitreous volume was 6.76 +/- 0.37 mm(3) in three weeks old rats and 13.36 +/- 0.64 mm(3) in two months old rats. Mean intravitreal ink volumes immediately after injection were 0.8 mm(3) for 1 ml injections, 2 mm( 3) for 3 ml, 2.3 to 2.6 mm(3) for 5 microl and 3.2 mm(3 ) for 10 microl. The percentage of vitreous volume involved by the injection ranged from 4.4% to 33.2%. The injected volume is limited by the large lens size of the rat. Extraocular loss of injected solution increases for higher injected volumes, with larger standard deviations. In this model, the dye tends to localize behind the lens. A 3 or 5 microl volume appears to have the best reproducibility with minimum loss of solution.

Aging↗
PubMed2006

Types and patterns of response in the larynx following inhalation.

The laryngeal mucosa responds to insult similarly to other epithelial tissues but the response depends on location within the larynx since important anatomic differences exist, even within rodent species. Although dogs and nonhuman primates are also utilized for inhalation toxicology studies, little published information is available regarding sites of injury from inhaled toxicants in these species. Accurate and consistent fixation, trimming, and microtomy of laryngeal sections allow the pathologist to compare the morphology of laryngeal mucosa from exposed and control animals and detect and interpret subtle differences resulting from inhalation exposure. There are anatomic landmarks that are keys to providing consistent sections through important areas of the laryngeal mucosa. Repeated inhalation of toxic concentrations of chemicals, drugs, or environmental contaminants induces a wide range of responses, depending on the physical properties and concentration of the toxic substance and duration of exposure. Responses include edema, acute to chronic inflammation, fibrosis, mucosal ulceration, degeneration, and necrosis. Attempts at repair include regeneration, hyperplasia, squamous metaplasia, hyperkeratosis, and neoplasia. Awareness of normal histology and zones of transition from squamous to respiratory epithelium in different areas of the larynx in different species is critical to avoid confusing normal epithelium with metaplasia or hyperplasia. Microscopic examination of laryngeal mucosa from animals exposed via inhalation and necropsied following a recovery period provides the opportunity to determine the degree of regression or progression of exposure-induced laryngeal lesions.

Animals↗
PubMed1995

DEFPARAM: a program package for aligning elliptical sections of biological objects containing an n-fold symmetry.

A program package has been developed to align automatically images of biological objects containing an n-fold symmetry, and to remove the distortions induced on their circular shape by the microtomy. It uses an original procedure based on correlation techniques and replaces usual manual processing. Examples of direct averaging of transverse sections of biological objects are given to illustrate the program's capabilities.

Algorithms↗
PubMed1989

Visual detection of microencapsulated insecticides with selective staining and scanning electron microscopy.

Selective staining with Sudan IV and methylene blue for light microscopy and scanning electron microscopy (SEM) were investigated to determine their potential for detecting and quantifying microencapsulated insecticides. Penncap-M (microencapsulated methyl parathion), Penncapthrin (microencapsulated permethrin), and Dyfonate (microencapsulated fonofos) were selectively stained with Sudan IV but not with methylene blue. Selective staining was not possible for Altosid SF-10 or SR-20 (microencapsulated methoprene) with either stain. Sudan IV enabled detection of some microencapsulated formulations in the digestive content of selected aquatic invertebrates and prepared contaminated pollen samples. Staining intensity with Sudan IV was greatest with acetone but capsular damage was high. A solvent ratio of 50:50 and 20:80 acetone/xylene minimized capsular collapse and maintained good staining intensity. The use of SEM for capsule identification and quantification depended upon the method of sample preparation: the slide smear method was superior to samples prepared by incision or microtomy. SEM was most suitable for investigation of formulations such as methoprene, for which selective staining was not possible. The chemical basis of staining with Sudan IV and potential application of both identification techniques are discussed.

Aedes↗
PubMed2002

Cervical spine lesions after road traffic accidents: a systematic review.

STUDY DESIGN: A systematic critical literature review. OBJECTIVES: To determine whether occult pathoanatomical lesions in the cervical spine of road traffic fatalities exist and if they can be identified using optimal autopsy techniques. SUMMARY OF BACKGROUND DATA: Previous investigations have examined pathoanatomical conditions of the cervical spine of road traffic fatalities. However, different methods of investigation have been used, and results of studies are conflicting. Hence, potential pathoanatomical conditions in fatalities and survivors remain a controversial issue. METHODS: Articles were retrieved searching the MEDLINE, Mantis, and Cochrane libraries. Studies examining the cervical spine of road traffic fatalities at autopsy were included and evaluated according to a set of quality criteria. For in-depth review, only studies using surface cryoplaning microtomy autopsy technique and a control group were included. RESULTS: Twenty-seven articles of which three fulfilled the quality criteria were reviewed. In these studies, subtle pathoanatomical lesions were found in the cervical intervertebral discs, cartilaginous endplates, and the articular surfaces and capsules of the zygapophysial joints. The lesions were found exclusively in the traumatized patients and in none of the patients in the control group. CONCLUSIONS: Occult pathoanatomical lesions in the cervical intervertebral disc and zygapophysial joints after fatal road traffic trauma may exist. Present imaging methods, especially conventional radiography, do not visualize these subtle lesions; hence, underreporting of pathoanatomical lesions during standard autopsy is probably common. These findings may have clinical relevance in the management of road traffic trauma survivors with potentially similar pathoanatomy.

Accidents, Traffic↗
PubMed2006

Plastic-embedded protein crystals.

Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.

Acrylic Resins↗
PubMed1980

A simple aid in the evaluation of glass knives.

This report describes a compact, portable, self-contained light-box for the qualitative inspection of glass knives under a microscope. It obviates the need for elaborate illumination and viewing facilities in the microtomy laboratory when the ultramicrotome is not itself equipped with knife-inspection devices.

Histological Techniques↗
PubMed1994

A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K.

Mixtures of ethanol, 2-propanol and 2-butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning. Brought directly from room temperature to a cutting support at 140 K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample. The mass hardens at lower temperatures fixing the sample well for microtomy. With ethanol: 2-propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K.

1-Propanol↗
PubMed1996

Surface and internal structure correlation: high-voltage and scanning electron microscopies of wholemount alveolar walls of human lung.

We found that the high-voltage electron microscope (HVEM) operating at 1.5MeV was able to transilluminate and form a focused transmission image of whole-mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph. Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features. Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 microns thick were delineated with the high-voltage electron microscope. Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other. HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.

Humans↗
PubMed1998

3D reconstruction of the mouse's mesonephros.

The present work reports on the three-dimensional reconstruction of the segmented mesonephros during the embryonic development of the mouse. With a light microscope and an automatic reconstruction of surfaces, aspects of the mesonephros are described. These surfaces are obtained by using digitized contour lines. A new interpolation method called DSI (Discrete Smooth Interpolation) enables correction of the distortion induced by microtomy in paraffin sections. After a triangulation step, this method uses a smoothing algorithm, which implies a spatial redistribution of the vertices of the triangles to correct the rotational and translational misalignment. The use of this 3D program improves the understanding of the development patterns and helps us to appreciate changes in the rebuilt mesonephros. By 10.5 embryonic days, tubules emerge from the Wolffian body and begin their formation, then between 11.5 and 13.5 embryonic days, tortuous mesonephric tubules bound to the Wolffian duct form small curls, which grow and finally unwind. At the same time, mesonephric tubules unbound to the Wolffian duct appear, and on 13.5 embryonic days, the Müllerian duct is visible. After 14.5 embryonic days, the segmented mesonephros keeps its general aspect but decreases in size. At this time, each gonad is provided with both Wolffian and Müllerian ducts. Later, the Wolffian duct differentiates into the definitive male duct system, whilst the Müllerian duct regresses. Conversely, the paramesonephric duct differentiates into the definitive female duct system, whilst the mesonephric duct in turn degenerates. By this time degeneration has begun in the cranial portion of the mesonephros and this process progresses caudally. The spatial organization of the mesonephric tubules and the precise organization of all connections between these elements and the ducts may be well defined. Such approach can allow for a high definition of the normal pattern of mesonephros differentiation.

Animals↗
PubMed1983

Oestrogen receptor assay of cryostat sections of human breast carcinomas with simultaneous quantitative histology.

Cryostat sections of unfixed human breast carcinomas were assayed for oestrogen receptor (ER) content using an isoelectric focusing method to separate the receptor-bound oestradiol. Adjacent sections from the same tissue block were stained so that the tumour content could be estimated by point counting and the ER concentration adjusted to compensate for variations in cellularity. Elastosis was also assessed. The results confirm a positive correlation between ER values and both cellularity and elastosis. The measurement of ER in cryostat sections is relatively simple and rapid, is applicable to small tissue samples, and permits histological identification of the nature and composition of the assayed sample. The method is directly applicable to oestrogen receptor analyses of breast carcinomas in clinical laboratories with facilities for cryostat microtomy.

Animals↗
PubMed1980

Quantitative microautoradiography of arteries: comparison of radioactivity to silver.

The local concentration of silver in developed aortic transmural microautoradiographs was compared to the corresponding 125I-labeled albumin radioactivity concentration [ci(x)] in the subjacent tissue. Silver [s(x)] was measured by electron probe X-ray microanalysis (EPA) and the corresponding ci(x) by direct gamma-ray counting. The results show 1) that the relationship between volume-averaged values of radioactivity (ci) and EPA signal (s) is adequately described by ci = Ks, where K is a proportionality constant, and 2) that ci(x) measured by EPA [i.e., Ks(x)] agrees closely with ci(s) measured direcly from en face microtomy slices of corresponding unfixed tissue specimens.

Animals↗