bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular distribution of actin in cells of the orgen of Corti: A structural basis for cell shape and motility
Authors: Slepecky Norma B. , Hozza Mark J. , Cefaratti Lisa
Published: February 2005

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post‐embedding staining of Lowicryl K4M sections, pre‐embedding staining of permeabilized cells of the organ of Corti, pre‐embedding staining of vibratome sections, and pre‐embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent—conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.
Analysis of neuronal networks: A review of techniques for labeling axonal projections
Authors: Ralston Henry J.
Published: February 2005

In order to analyze connections between neurons in the vetebrate central nervous system, methods have been developed to label a given population of axons of known origin so that they can be differentiated from other, non‐labeled structures. Three such methods are reviewed here: experimentally induced orthograde (Wallerian) degeneration, axon transport of radioactive proteins demonstrated by autoradiography, and axon transport of macromolecules that can be reacted histochemically to yield a visible reaction product. Each of the methods has particular strengths and weaknesses. Degeneration methods may differentiate between different functional classes of axons which have different fiber diameters. However, degeneration distorts the morphology of axon terminals, making them more difficult to interpret, and degenerating terminals may be removed rapidly by phagocytosis. Autoradiography of radioactive terminals preserves normal fine structure, but the necessary exposure times extend the method by weeks or months, and care must be exercised to distinguish labeled axons from other structures exhibiting background or transneuronal radioactivity. Histochemical methods, such as those used to demonstrate horseradish peroxidase conjugated to wheat germ lectin (WGA‐HRP), are sensitive and rapid, but the injection site must be carefully characterized, and the presence of transneuronal label may make interpretation of the results difficult.

Experimental methods of axonal labeling have been invaluable in studying neuronal networks. Each of the methods described here may be of particular value, given the nature of the system to be analyzed.
Synaptic connections of neurones identified by Golgi impregnation: Characterization by immunocytochemical, enzyme histochemical, and degeneration methods
Authors: Somogyi Peter
Published: February 2005

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi‐impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi‐impregnated neurones and their pre‐and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre‐and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP‐filled neurones and Golgi‐impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.
Preparation of fetal rat brains for light and electron microscopy
Authors: Åström Karl E. , Webster Henry deF.
Published: February 2005

To study cellular shapes, growth patterns, and fine structure during early stages of CNS development in rat embryos, preparative procedures were evaluated and modified to meet two criteria: (1) Coronal semithin sections should reveal undeformed telencephalic hemispheres that were symmetrically expanded on both sides of midline structures and were surrounded by contiguous mesenchyme. (2) In electron micrographs, cells should have intact, undistorted surface membranes, evenly distributed nucleoplasm and well preserved cytoplasmic organelles. To meet these criteria, 378 fetuses with a gestational age of 11–20 days (E11–E20) were used to test and modify procedures for anesthesia, embryo removal and handling, dissection, fixation, dehydration, and embedding of the embryonic CNS. Most specimens were in an early stage of development (E11–E13), which, in case of the neopallial wall, is the preneural period. The tests produced methods that met the above criteria and identified the most common artifacts and their causes. Deformities of the cerebral hemispheres and separations between the brain and its coverings were usually caused by trauma during embryo removal and during handling before fixation. Changes in cellular volumes, especially swelling during fixation and dehydration, were the most important causes of histological artifacts. The procedures and methods that consistently produced the best light and electron microscopic preservation of the E11–E13 rat CNS are described. Fixation was best when the brains were treated with glutaraldehyde and s‐collidine buffer, followed by osmium tetroxide in s‐collidine buffer. A surprisingly beneficial effect of sodium chloride in the dehydrating alcohol was noted.
Fasting induces modifications of the endoplasmic reticulum in intestinal cells
Authors: McLeese Jennifer , Bergeron Michel
Published: February 2005

The polymorphism of the endoplasmic reticulum (ER) in epithelial cells with different transport functions such as the enterocyte suggests that the ER may be involved in some way in molecular transport. To further access this possibility, we examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes an annual fast of approximately 6 months' duration, a time during which previous work indicates nutrient carrier number does not change. Fish from June (feeding) and January (8–10 weeks fasted) were sampled. Tissues from the pyloric caeca, foregut, midgut, and hindgut were prepared for electron microscopy using two techniques of staining. Cell height was unaltered in any section, although microvillar length shortened variably. Cellular organization, including position of nuclei, number and distribution of mitochondria, and presence of basolateral membranes, did not change. The ER appeared equally abundant in June and January. However, use of the osmium impregnation technique, which is specific for ER cisternal contents, revealed a change in the impregnation of ER, from a heavily impregnated network in summer to little or no impregnation in winter. These results suggest that a shift in function of the ER had occurred when nutrient transport ceased, and supports a role of the ER in nutrient transport.
Cellular and developmental biological aspects of bovine meiotic maturation, fertilization, and preimplantation embryogenesis in vitro
Authors: Van Blerkom Jonathan , Bell Hobart , Weipz Deborah
Published: February 2005

Cellular aspects of reinitiated meiosis, fertilization, and early preimplantation embryogenesis in the bovine species were examined under in vitro conditions. An analysis of the cytoplasmic distribution of mitochondria, lipid droplets and vesicles in over 5,000 living GV‐stage oocytes, with subsequent examination by electron microscopy, indicated that the organization of cytoplasm was pleomorphic and that five distinct cellular phenotypes could be identified. Inspection of oocytes during the resumption of arrested meiosis in vivo and in vitro demonstrated that the subcellular organization of the bovine oocyte cytoplasm remains unchanged during maturation to metaphase II. The influence of culture conditions and media on the frequency of maturation, cytoplasmic organization, fertilizability, and early preimplantation embryogenesis was also determined. The findings indicate that meiotic maturation and fertilization in the bovine species can occur at high frequency under comparatively simple and defined conditions. However, the acquisition of developmental competence for fertilization, and the ability of the egg to develop progressively after fertilization appears to be related to the organization of the cytoplasm at the GV stage. The relationship between cytoplasmic organization and conditions of maturation, fertilization, presence or absence of cumulus cells, and the acquisition of developmental competence is discussed with respect to (1) cell biological aspects of mammalian oocyte maturation, (2) the potential influence of extrinsic factors (e. g., differential intrafollicular biochemistry and morphophysiology) on sub‐cellular organization of the GV‐stage oocyte, and (3) the finding that morphologically equivalent bovine embryos derived from the in vitro fertilization of in vitro‐matured oocytes may be developmentally heterogeneous. The studies also revealed that nuclear and cytoplasmic aberrations which could preclude normal embryogenesis can develop shortly after fertilization. The significance of this finding with respect to cytoplasmic phenotype of the oocyte and conditions of maturation, fertilization, and early embryo culture is discussed.
Real space modulation in bi2Sr2CanCun+1O6+2n and TI2Ba2CuO6 Superconductors Derived From Electron Diffraction Information
Authors: Verwerft Marc , Tendeloo Gustaaf Van
Published: February 2005

We will try to illustrate here that, simply from the geometry of the electron diffraction pattern of an incommensurably modulated structure, conclusive information can be obtained on the real space shape of this modulation. The method applied here is based on the 3 + 1 dimensional description of symmetry operations and can be summarized as follows: (1) reconstruct the three‐dimensional reciprocal space geometry of the modulated structure from electron diffraction information along different zone axes; (2) deduce the complete Bravais type symbol of the four‐dimensional structure from the general reflection conditions; and (3) derive the modulation function for each atom type from the superspace symmetry elements which result from the information of both modulation and basic structure. This method will be applied here in short on the Bi2Sr2CanCun+1O6+2n structure, for which system the results are in agreement with the ones recently obtained from neutron diffraction. For Tl2Ba2CuO6 where no data from other diffraction techniques are available, a more complete calculation will be performed, in order to determine the shape of the displacement function for the different atom types; the results are in agreement with the observed High Resolution Electron Microscopy (HREM) images.
Microwave energy fixation of plant tissue: An alternative approach that provides excellent preservation of ultrastructure and antigenicity
Authors: Benhamou Nicole , Noel Sylvain , Grenier Jean , Asselin Alain
Published: February 2005

Immunocytochemical techniques are confronted with the problem of obtaining adequate tissue preservation together with retention of protein antigenicity. Various methods, including freeze‐drying and freeze‐substitution, have been devised to circumvent this problem. In the present study, we report that microwave energy used in combination with low concentrations of glutaraldehyde (0.1%) and paraformaldehyde (2%) preserves the structural integrity of plant tissue and antigenicity of proteins. Tobacco leaf samples fixed in a time as brief as 15–20 s exhibited excellent preservation of fine structures. By contrast, specimens irradiated for shorter (5–10 s) or longer (30–40 s) periods showed poor morphological preservation. Microwave irradiation for 15–20 s was found useful for immobilizing large amounts of soluble antigens. The fast microwave fixation method was successfully used to preserve pathogenesis‐related (PR) proteins, which were subsequently localized by a postembedding immunogold procedure. In addition to soluble antigens, cellulose subunits and pectic substances, two major plant cell wall components, were found to be highly preserved in microwave‐irradiated tobacco plant tissue. The present study demonstrates that microwave fixation of plant tissue is a simple and inexpensive method that is easy to perform with commercially available microwave ovens. The incubation time for fixation is reduced from 2 h to 15–20 s without loss of fine structural details. This method will undoubtedly acquire increasing applicability and relevance in plant biology.
Extending the limit of atomic level grain boundary structure imaging using high‐resolution electron microscopy
Authors: Krakow William
Published: February 2005

It has been possible to image ∑ = 21/[111]21.8° tilt boundaries in thin Au films and to deduce their atomic arrangements. These results represent an electron microscopic resolution level of 1.43 Å, attainable with a small amount of image processing, which produces interpretable structure images. This substantial improvement over other recent grain boundary studies, which required about 1.9–2.0 Å resolution, clearly demonstrates that many more tilt grain boundary orientations are now accessible instead of a limited subset.
Transmission electron microscope in situ fatigue experiments: A computer‐control approach
Authors: Vecchio Kenneth S. , Hunt John A. , Williams David B.
Published: February 2005

A computer‐control procedure was developed to facilitate in situ fatigue experiments within an intermediate voltage transmission electron microscope using a goniometer‐type straining holder. The procedure was designed to allow sine‐wave tension‐tension cyclic loading of a microfatigue specimen similar in geometry to a center‐crack panel fatigue specimen. Computer control allows greater freedom for the operator to control the experiments while providing better reproducibility from one test to another. Further development of this procedure is possible by coupling this computer‐control technique with computer‐controlled stage motion and digitized TV imaging.
Oxygen content of the fixative is important in the interpretation of the ultrastructure of ischaemic myocardium
Authors: Maxwell Linda , Gavin John Bevan , Walker Sharon
Published: February 2005

Isolated rat hearts were subjected to 15, 45, or 60 minutes of global ischaemia and then fixed by perfusion at 37°C with glutaraldehyde containing various amounts of oxygen. This either had been bubbled with 100% oxygen (PO2 620 mm Hg) or with 100% nitrogen (PO2 40 mm Hg) immediately before use, or it had been routinely prepared and stored exposed to atmospheric oxygen (PO2 245 mm Hg). The ultrastructure of myocytes and endothelial cells subjected to 15 minutes of ischaemia was not affected by the treatment of the fixative. However, when the tissue subjected to longer periods of ischaemia was fixed with routinely prepared or oxygen‐bubbled glutaraldehyde, ultrastructural changes characteristic of reoxygenation damage were uniformly evident in both the microvasculature and myocytes. These qualitatively distinct changes included mitochondrial swelling, cell swelling, endothelial bleb formation, and narrowing of capillary lumina. These abnormalities were not observed in tissue fixed with nitrogen‐bubbled glutaraldehyde. These findings indicate that deliberate steps should be taken to reduce or eliminate dissolved oxygen from the fixatives used to study ischaemic tissues. Otherwise artefactual reoxygenation damage in vitro may occur and make valid ultrastructural interpretation difficult or impossible.
Physiology and pathophysiology of the human spermatozoon: The role of electron microscopy
Authors: Zamboni Luciano
Published: February 2005

In this article, the major contributions of electron microscopy to the present understanding of the physiology and pathophysiology of the human spermatozoon are reviewed. The ultrastructural organization of sperm organelles playing a significant role for cell function and, therefore, for the reproductive process is described. Also, the major abnormalities and defects of the various organellar systems and how they impair the reproductive function and/or the viability of the cell are reviewed.
Dispermic fertilization of human oocytes
Authors: van Blerkom Jonathan , Henry George
Published: February 2005

Regardless of whether fertilization occurs in vivo or in vitro, polyspermic penetration of the human oocyte is a not infrequent cause of reproductive failure. This report describes the occurrence of human eggs fertilized by two spermatozoa and the variable developmental potentials expressed by the resultant embryos. The cellular and subcellular events that characterize the pronuclear and early cleavage stages of preimplantation embryogenesis are discussed with respect to the ability of such embryos to progress to implantation.
Versatile controlling system for cryopreparation techniques in electron microscopy
Authors: Humbel Bruno M. , Weber Klaus , Merkl Rainer
Published: February 2005

A new setup for freeze‐substitution and a versatile controlling system has been developed. Our goal was to build a simple system allowing precise control of the physical parameters of freeze‐substitution experiments to learn more about their influences on the cellular ultrastructure and immunoreactivity of macromolecules. An improved apparatus for freeze‐substitution, based on liquid nitrogen cooling, and a universal software for controlling the complex preparation protocols from cryofixation to final polymerization are described. This controlling system has the following advantages: it allows precise control and registration of temperature profiles, reconstruction of each individual step of previous experiments, and optimization of working conditions. The setup of the freeze‐substitution apparatus is designed to run many different substitution media in parallel; freeze‐substitution (cryostat), embedding (working platform), and polymerization are carried out at separate places; therefore, more experiments can be done simultaneously. The ergonomic working platform allows exchange of media at controlled temperature and easy handling; survey of the temperature in individual tubes is possible, and the system is protected from water condensation and uncontrolled warming by the deep freezer.
Determination of vesicle size distributions by freeze‐fracture electron microscopy
Authors: Hallett F. R. , Nickel B. , Samuels C. , Krygsman P. H.
Published: February 2005

The most common electron microscopic technique for obtaining information on size distributions of uncollapsed membrane vesicles is based on the method of van Venetie (1980). This technique involves the sizing of only those vesicles that were freeze fractured at their equatorial planes. As a result, only a small number of images can be used to generate size distributions. Further, the technique is susceptible to systematic error. An alternate approach is to consider the complete distribution of image sizes and use this distribution to determine the average size and distribution of the vesicles. It is shown that the mean vesicle size is 4/π times the mean image size. As well, a parameter, m, which can be determined from the image distribution, can be used to characterize the vesicle distribution. The advantage of this new approach is that images of all vesicles are used, leading to a statistically better determination of vesicle sizes.
In vivo, real‐time confocal imaging
Authors: Jester James V. , Andrews Peter M. , Matthew Petroll W. , Lemp Michael A. , Dwight Cavanagh H.
Published: February 2005

We have adapted a tandem scanning confocal microscope for real‐time, non‐invasive imaging of cells under in vivo conditions. This form of in vivo confocal imaging relies on the optical sectioning abilities of the confocal microscope to obtain en face, sequential, reflected light images of cells at various depths, up to 1 mm, within opaque organs in living animals. Of major consideration in the design of an in vivo confocal microscope is maximizing the real‐time detection of signals reflected from low contrast structures which can be affected by the microscope design, objective, and image detector systems. Using an in vivo confocal microscope design with a 20 × BioOptics surface contact objective we have obtained live cellular images from selected tissues including cornea, kidney, liver, adrenal, thyroid, epididymis, and muscle and connective tissue of rabbits and rats. Images were captured, digitized, and processed using a DAGE Mti low light level SIT camera coupled to a Gould IP9527 image processor. In vivo images were also compared with conventional bright field light and scanning electron microscopic images of “dead,” fixed tissues. Overall, in vivo confocal imaging can provide remarkable detail of living cells comparable to that of conventional microscopic images of “dead,” fixed, and stained tissue. A more unique feature of in vivo confocal imaging is the ability to study cellular structure and function sequentially over time in the same organ or tissue and represents a fundamentally new paradigm in microscopy. With continued refinements in the microscope, objective and detection system designs and their consequent improvements in lateral and axial resolution, in vivo confocal microscopy will enable us as observers to see what no one has been able to see before.
Ultrastructural investigation of neurons identified and localized using the confocal scanning laser microscope
Authors: Deitch Jeffrey S. , Smith Karen L. , Swann John W. , Turner James N.
Published: February 2005

Studies of labeled neurons at the light‐microscopic level often pinpoint a substructure of particular interest, i.e., a synapse or a spine. An ultrastructural investigation would explain a lot about how these structures arose, how they function, and how they are regulated. Finding a small region in a large block can require constant checking during sectioning, until past the structure. In our pursuit of the synaptic structure of varicosities on the axons of neurons identified physiologically and morphologically at the light level, we have combined confocal scanning laser microscopy (CSLM) with conventional and high‐voltage electron microscopy (EM). CSLM images were collected in the reflection mode to view neurons filled with horseradish peroxidase and stained with nickel‐intensified diaminobenzidine, which is compatible with EM. The CSLM optical sections provided a record of what one should expect to see at regular intervals throughout the depth of the tissue block. We have shown that the CSLM greatly simplified the task of localizing small structures in brain tissue prepared for EM.
Immunogold labelling of the intermediate filament‐lamina‐nuclear matrix system in hela and bhk‐21 cells
Authors: Jiao Renjie , Wu Donglan , Zhang Bo , Cai Shutao , Zhai Zhonghe
Published: February 2005

Whole‐mount, sequentially extracted cells combined with immunogold electron microscopy were developed to demonstrate the intermediate filaments, lamina, and nuclear matrix (IF‐L‐NM) and to identify their protein components. The IFs of HeLa cells were reacted both with keratin and vimentin monoclonal antibodies; meanwhile, the IF network of BHK‐21 cell was reacted only with vimentin monoclonal antibody. The lamina and nuclear matrices of both HeLa and BHK‐21 cell were labelled, respectively, with lamin monoclonal antibody‐gold complex and 280 Kd nuclear matrix protein monoclonal antibody‐gold complex. The monoclonal antibody to keratin could cross‐react with the lamina both of HeLa and BHK‐21 cells.
Cellular location of thymus‐leukemia (TL) antigen as shown by immuno‐cryoultramicrotomy
Authors: Hartfield Cheryl , McDowall Alasdair , Loveland Bruce , Lindahl Kirsten Fischer
Published: February 2005

Thymus‐leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno‐cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti‐TL antibody and protein A‐gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one‐step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitochondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.
Immunoelectron localization of mitochondrial F1 factor in cryosections of heart muscle cells
Authors: Real Suzana Corte , de Meirelles Maria de Nazareth Leal
Published: February 2005

Immunocytochemistry was used to investigate the localization of F1 ATPase in mitochondria of cryosections of adult mouse heart muscle cells. The initial aldehyde fixation was the only denaturation step for antigens. The fine structure was preserved with contrast enhancement as the sections were maintained hydrated, with the advantage that the entire procedure is completed in one working day. The reaction was highly specific, and entire mitochondria were labeled with the Protein A‐gold complex. A new analytical technique, electron spectroscopic imaging (ESI), contributed to a better visualization of the localization of the F1 factor.
Ultrastructural localization of gold particles within neural grafts labeled with gold‐filled sendai viral envelopes
Authors: Kott Jon N. , Westrum Lesnick E. , Ardizzoni Susan C. , Arendash Gary W.
Published: February 2005

The ability to discriminate between host and donor cells is required to interpret the organization of neural grafts at the electron microscopic (EM) level. Using light microscopy, Ardizzoni et al. (Ardizzoni, S.C., Michaels A., and Arendash, G.W. [1988] Science 239: 635–637) described a method, using gold‐filled Sendai viral envelopes, for labeling cell suspensions prior to grafting. As the colloidal gold used in this procedure is especially attractive for use with EM, we have examined the ultrastructural distribution and character of this label with transplanted cells. Cell suspensions taken from the nucleus basalis of fetal rats were labeled using gold‐filled Sendai viral envelopes and grafted into the dorsal neocortex of adult host rats with nucleus basalis lesions. After varying survival times ranging from 1 to 14 months, grafts and surrounding host tissue were examined using standard EM techniques. Within the graft site, gold particles ranging from 10–200 nm were found associated with various membranes throughout the cytoplasm of both neurons and glia. Gold particles of similar size were also found within the nuclei of neuronal and non‐neuronal cells. Host cells near the graft site contained some small gold particles (10–40 nm). Control injections of non‐viable, gold‐labeled cells or colloidal gold alone resulted in similar patterns of small gold particles which were readily discriminable from the larger virally inserted gold particles found in viable labeled donor cells. We conclude that this method allows discrimination between closely associated host and donor cells.
A study of protein A‐gold resolution for immunoelectron microscopy
Authors: Shida Hisato
Published: February 2005

For the purpose of investigating a topographical correlation between antigen molecules and protein A‐gold(PAG) particles which localized as an immunocytochemical probe, the simplest model on a localization pattern of antigen molecules, which were arranged two‐dimensionally on a plane surface of the resin, was used. Ultrathin sections of a G‐actin layer, which was adsorbed on epoxy resin and was re‐embedded subsequently in JB‐4 resin, was stained indirectly with rabbit anti‐actin antibody and subsequently by PAG. From this immunoelectron microscopy, a histogram (relative frequency, denoted by y vs. relative length, denoted by x) was obtained using a computer‐assisted method. For this histogram, a fitting curve was calculated by a least squares optimization and three parameters (H, U, and W) of the curve which could be useful for a study on the topographical organization of antigen molecules were estimated. Parameter H (maximum y of the curve) would reflect the maximum amount of epitopes at x = U. Half width W, which is the width of the curve at y = H/2, would reflect a breath of epitope masses. This fitting curve was separated into two overlapping curves whose Ws were different from each other. The one constituent curve of which value W was smaller than the other was regarded as a unit curve and the other constituent curve could be resolved into many unit curves whose W values are the same. From these unit curves, the resolution power of the immunoelectron microscopy, using a post‐embedding procedure of ultrathin sections, was estimated as 58–66 A°.
Similar features in Z bands of both skeletal and cardiac muscle revealed by image enhancement
Authors: Schroeter John P. , Bretaudiere J. P. , Goldstein Margaret A.
Published: February 2005

We have shown previously that the small square (ss) and basket weave (bw) states of the Z band lattice in cardiac and skeletal muscle are related to the contractile state of the muscle. We have used two‐dimensional image processing techniques on digitized electron micrographs to enhance the structural features of each projected lattice form in cardiac and skeletal muscle. Four different processing techniques were employed to assess the effect of enhancement artifacts on the resulting Z band images.

We observed only slight differences between enhanced images of a particular Z band form produced by the four different techniques. Every enhanced image showed an approximate four‐fold symmetry independent of muscle type or Z band lattice form. Each enhanced image showed four cross‐connecting Z‐filaments which appeared to connect each axial filament to the four nearest axial filaments. In bw images from both cardiac and skeletal muscle, axial filaments had a greater apparent diameter and a greater interaxial filament spacing than in the ss images. In both muscle types, the cross‐connecting Z‐filaments appeared to overlap half‐way between axial filaments in the ss images while the bw images showed no such overlap. These structural features are consistent with a dynamic Z band lattice that participates in muscle contraction.
A technique for the preparation of cross‐sectional TEM samples of ZnSe/GaAs heterostructures which eliminates process‐induced defects
Authors: Yu J. E. , Jones K. S. , Park R. M.
Published: February 2005

Cross‐sectional transmission electron microscopy (TEM) sample preparation of ZnSe/GaAs epitaxial films is investigated. Conventional argon ion milling is shown to produce a high density (∼ 5–8 × 1011/cm2) of small (diameter ∼ 60–80 Å) extended defects (stacking faults, microtwins, double positioning twins, etc.). In addition, transmission electron diffraction results indicate a thin ZnO layer can also occasionally form upon ion milling or electron‐beam irradiation although the exact conditions for ZnO formation are not well understood. Conventional TEM (amplitude contrast) and high‐resolution TEM (phase contrast) imaging in combination with transmission electron diffraction studies were performed to determine the optimum method of removing the ion milling related damage and ZnO layers during sample preparation. HF/HCl, NaOH/H2O, H2SO4/H2O2/H2O and Br2/CH3OH etching mixtures as well as low voltage argon or iodine ion milling were studied. A low energy (2 ke V) iodine or argon ion milling step was shown to remove the ZnO layer and reduced the density of the extended defects associated with Ar+ ion milling, but was unsuccessful in removing all of the defects. Auger electron spectroscopy results indicate residual iodine was either left on the surface or implanted beneath the surface during iodine ion milling. Etching the XTEM samples in HF/HCl was shown to be effective in removing the ZnO layer but had little or no effect on the ion milling induced defects. Etching the samples in a 0.5% Br2/CH3OH solution resulted in complete elimination of the ion milling induced extended defects including the residual defects associated with iodine ion milling. In addition the Br2/CH3OH etch produced the best surface morphology. Thus a brief (1–2 seconds) Br2/CH3OH etch after conventional preparation (argon ion milling) of cross‐sectional ZnSe/GaAs TEM samples appears to be an inexpensive and superior alternative to iodine ion milling.
Simple method for the preparation of inp based samples for TEM investigation
Authors: Barna Árpád , Pécz Béla
Published: February 2005

A novel, rapid, and simple method is described for the preparation of InP based samples for investigation by transmission electron microscopy (TEM). The key feature of the technique is Ar+ ion bombardment in an iodine ambient. Cross sectional micrographs of Au/InP samples are shown as an example. The technique developed produces a large area of transparent region.
Fetal development and regulation of pituitary cell types
Authors: Dubois Paul M. , Hemming Fiona J.
Published: February 2005

The ontogenesis of the pituitary gland is considered from anatomical and functional points of view. Embryogenesis of the hypothalamo‐pituitary unit involving development of the hypothalamo‐hypophyseal portal system is complete during early life as shown in several mammalian species. The ultrastructural characteristics of the different cell types during development are described according to observations made by using immunochemical techniques. The patterns of differentiation of the cell types are reviewed according to studies of pituitary glands from human anencephalic fetuses and encephalectomized rat fetuses as well as in vitro studies of cultured pituitary primordia in synthetic media. The maturation of the neuroendocrine mechanisms controlling the secretion of fetal hormones is also analyzed. During fetal life, the factors implicated in the regulation of pituitary hormone secretion are generally the same as in adults, but the intensity of the response of pituitary cells to their action is variable according to the species, thus reflecting an immaturity in the functioning of certain cell types.
Application of preembedding immunohistochemistry in the study of adenohypophysial plurihormonality
Authors: Osamura R. Yoshiyuki
Published: February 2005

An immunoelectron microscopy preembedding technique has been utilized as a method which enables the observation of various bioactive substances in various intracellular organelles. In this review article, the observations made by preembedding immunoelectron microscopy describe plurihormonal pituitary cells emphasizing subcellular localization of hormones and their alterations.
Ultrastructure of the aging human testis
Authors: Paniagua Ricardo , Nistal Manuel , Sáez Francisco J. , Fraile Benito
Published: February 2005

The ultrastructure of the progressive testicular involution with advancing age in men is reviewed. There is no definite age at which testicular involution begins, and the onset and severity of testicular lesions are subjected to pronounced individual variations. Hormone studies also indicate great individual variations, and subtle changes in both the testis and the pituitary develop progressively with age. Testicular size, sperm quality, and numbers of all germ cell types, Sertoli cells, and Ley dig cells decrease with age. The volume occupied by the seminiferous tubules decreases, whereas that occupied by the testicular interstitium remains constant. The most frequent histological pattern of the aging testis is a mosaic of different seminiferous tubule lesions, varying from tubules with complete, although reduced, spermatogenesis, to completely sclerosed tubules. The tubules with complete spermatogenesis may show numerous morphological abnormalities in the germ cells, including multinucleation. Abnormal germ cells degenerate causing Sertoli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytosed by the Sertoli cells give rise to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with the spermatids, but progressively affects the earlier germ cell types, and tubules with maturation arrest at the level of the spermatocytes or spermatogonia are observed. The Sertoli cells show morphological abnormalities such as dedifferentiation, mitochondrial metaplasia, and multinucleation. Germ cell loss is associated with thickening of the tunica propria. When all seminiferous epithelial cells have disappeared, only an intensely collagenized tunica propria with myoid cells remains (sclerosed tubules). The Ley dig cells progressively dedifferentiate with a decrease in the quantity of both smooth endoplasmic reticulum and mitochondria, together with an accumulation of lipid droplets, crystalline inclusions, and residual bodies, and formation of multinucleate cells. The development of tubular involution with age is similar to that observed after exprimental ischemia, suggesting that vascular lesions may play an important role in age‐related testicular atrophy.
Immunogold electron microscopic localization of antigenic sites in the outer dense fiber region of rat sperm tail obtained by using antisera to two Sertoli cell secretory proteins
Authors: Kierszenbaum Abraham L. , Ueda Hiroshi , Tres Laura L.
Published: February 2005

Polyclonal antisera raised against polypeptide components of two rat Sertoli cell secretory proteins, designated protein S70 and S45–S35 heterodimeric protein on the basis of cell origin and estimated molecular weight, were used to identify antigenic sites in (1) rat testis, () cultured Sertoli cells, (3) developing spermatids (collected from spermatogenic stage‐specific seminiferous tubular segments), and (4) epididymal sperm. Indirect immunofluorescence, immunoper‐oxidase, and immunogold electron microscopy (single and double labeling) were used. Immunocytochemical techniques have detected antigenic sites in (1) the cytoplasm of Sertoli cells in the intact seminiferous tubule and in culture in the form of a punctuate, granular‐like pattern, and (2) the acrosome (but not the Golgi region) and tail of developing spermatids and sperm. In developing spermatids, the principal piece of the tail displays a characteristic apical‐to‐distal immunoreactive banding pattern that correlates both temporally and spatially with the reported multistep assembly of outer dense fibers along the axoneme. The immunoreactivity of the acrosome, connecting piece, and outer dense fibers of the sperm tail was confirmed by immunogold electron microscopy. A precise identification of the component(s) of the outer dense fiber region responsible for the antigenic homology with Sertoli cell secretory proteins is under investigation.
Cryofixation of vascular endothelium
Authors: Wagner Roger C. , Andrews S. Brian
Published: February 2005

Cryofixation refers to the immobilization of tissue components by the rapid removal of heat from the specimen, so that the structure is interred and stabilized in a natural embedding medium, namely, frozen (amorphous or microcrystalline) tissue water. Cryofixation is now often used as a complement to the more traditional fixation methods, especially when the cell structure is delicate or dynamic and may be inaccurately preserved by the slow selective action of chemical fixatives. Vascular endothelial cells are specialized for transcellular transport and for the regulation of blood flow and composition. The dynamic and labile subcellular organization of these cells, presumably reflecting these functional specializations, makes them ideal candidates for cryofixation.

Several different types of endothelial cells were directly frozen at temperatures below 20 degrees Kelvin by pressing them against a liquid‐helium‐cooled block. These samples were subsequently processed for structural analysis by freeze‐substitution. Detailed rationales, designs, and protocols are described for both freezing and freeze‐substitution.

Electron micrographs of cryofixed arterial and venous capillaries (rete mirabile of the American eel), iliac vein (rabbit), and cultured endothelium from the iliac vein (human) reveal that the organization of the characteristic intracellular membrane system of endothelial vesicles is qualitatively similar to that seen in chemically fixed endothelium, especially with regard to the interconnection of clusters of individual vesicles to form elaborate networks. The luminal and abluminal networks are not in communication, at least not in static images. Quantitatively, however, most directly frozen endothelial cells have far fewer vesicular profiles than comparable glutaraldehydefixed cells. The differences can be explained by presuming that the rapid action of cryofixation (approximately 1 msec) gives a more accurate picture of the vesicular network because it captures the transient structure of labile or dynamic membranes.
The endothelial vesicle system in cryofixed frog mesenteric capillaries analysed by ultrathin serial sectioning
Authors: Frøkjæer‐Jensen Jørgen
Published: February 2005

Conventional EM sections of chemically fixed capillary endothelial cells reveal numerous apparently free smooth plasmalemmal vesicles. However, the method of ultrathin (< 150 Å) serial sectioning has shown that the smooth vesicle profiles arise merely as a result of the EM thin sectioning of two sets of complex vesicular invaginations from the luminal and abluminal cell surfaces, which end blindly in the cytoplasm. While 50–70% of the total population of vesicular profiles appear to lack connections to the cell surface in conventional (500–700 Å thick) EM thin sections less than 1% truly free vesicles can be found by the ultrathin serial section analyses.

In the present study it is examined whether similar conclusions apply to endothelial cells which were directly frozen by slam‐freezing and subsequently freeze‐substituted. The three‐dimensional organization of the plasmalemmal vesicular system was analyzed in four series of 19, 18, 13, and 10 ultrathin sections (approximately 110 Å thick) of capillaries from frog mesenteries quickly excised from decapitated frogs (Rana pipiens). None of 920 vesicular profiles (diameter 500–1,200 Å) which appeared free in individual thin sections of the series represented free vesicles; all profiles either communicated with other vesicles, the cell surface, or in rare cases turned out to be part of cytoplasmic tubular membrane structures. It is concluded that free smooth plasmalemmal vesicles are very rare in rapidly frozen as well as in directly fixed frog capillary endothelium.

The volume density of profiles (13–15%), the proportion of apparently free vesicle profiles (70%), and interconnected profiles (20%) were similar to the picture previously found in single EM sections of frog mesenteric capillaries. No transendothelial channels were found in the four series of ultrathin sections of capillaries. However, continuities between the luminal and abluminal cell surfaces were seen in the endothelium of venules. Furthermore, in the ultrathin series of the capillaries, vesicular units belonging to the two sets of invaginations and cytoplasmic tubular membrane structures were in more cases found in very close contact—as fused to share one unit membrane. If this finding is representative for the in vivo situation, it may reflect that the vesicular system represents a highly dynamic system with possibilities for mixing of membranes, cellular traffic of lipid, membrane proteins, and receptors between internal compartments and the cell surfaces, as well as occasional exchange of macromolecules between blood and tissue through rare temporary connections between the two sets of surface invaginations, without actually moving vesicles.
Lectin and immunolabeling of microvascular endothelia
Authors: Milici Anthony J. , Porter Glyn A.
Published: February 2005

A number of recently developed localization techniques are beginning to be applied in the study of endothelial cells and their structural components. In this article we will review a number of these cytochemical approaches as well as their advantages and disadvantages and their applications. The methods will be presented for processing tissues for either L.R. White embedding or semi‐thin and thin frozen sections followed by subsequent lectin and immunolabeling for fluorescence and electron microscopic examination. These techniques are easily applied in the localization of perfused exogenous proteins and of endogenous endothelial‐associated proteins. The results that can be obtained from such studies are presented and discussed.
Quick‐freeze, deep‐etch studies of endothelial components, with special reference to cytoskeletons and vesicle structures
Authors: Izumi Toshihiro , Shibata Yosaburo , Yamamoto Torao
Published: February 2005

A three‐dimensional study of the ultrastructure of endothelial cells is helpful in understanding important endothelial functions such as vascular transport and cell permeability. For this purpose, in addition to serial sectioning electron microscopy and high‐voltage electron microscopy, the quick‐freeze, deep‐etching technique also enables us to analyze structures at the molecular level by its high resolution and is useful for three‐dimensional morphological studies.

Some modifications on the conventional deep‐etching method were made in this study to reduce the undesirable aggregation of proteins and salts during etching. Using this technique, we examined the rat aortic endothelium, particularly the membrane structures and cytoskeletons.

The luminal surface of the endothelium was covered with a fine filamentous coat, which was anchored to the plasma membrane. In the cytoplasm, actin filaments were prominent and were oriented randomly or in a parallel fashion near the plasma membrane. Of the vesicles seen in the endothelium, some had basket coats of clathrin, and others had striped coats on the cytoplasmic membrane surface. These surface structures of the vesicles suggest the transport mechanism of the vesicles in association with the fine filaments attached to the vesicles.