Brush border myosin I (BBMI) was first identified as a major constituent of the microvillus cytoskeleton, where it links the actin filaments of the microvillus core to the overlying plasma membrane. Recently, however, BBMI was also found on isolated Golgi membranes. This observation suggests that cytoplasmic BBMI might act as a molecular motor for apically targeted secretory vesicles.

In an earlier study from this laboratory a 105-110 kD protein was isolated from the chicken small intestine, which displayed several characteristics of the BBMI. Using an antibody against this BBMI- like protein (MLP), the protein was detected in the apical brush border of the enterocytes. The limited resolution of the light microscopic immunohistochemical techniques used in that investigation, however, did not provide information about the intracellular distribution of MLP in the enterocytes.

In this study the intracellular localization of MLP was characterized using post-embedding colloidal gold immunocytochemistry. Pieces of the jejunum from 6-week-old male White Leghorn chickens were fixed and embedded in LR Gold resin.

We developed a new type 3-Dimensional Internal Structure Microscope (3D-ISM) for the observation of internal structures of samples. The internal structure of a sample is obtained by cutting it into thin slices and observing the cutting side continuously while cutting. The position of the camera, as well as the sample position are fixed. Therefore there is no shift between the sections, and this system can obtain a true color image of the sample, which is a high resolution and a high-quality three-dimensional image compared with X-ray CT and MRI. After repeatedly slicing a sample, the digital image data from the sectional views is transferred to a computer, where 3-dimensional images of the internal structure of the sample are reconstructed. Using this system we analyzed many biological organisms. In this paper, a mouse specimen has been cut and the 3-dimensional images are shown.

This article presents the outline of the device and the principle of the observation. FIG.l shows a corresponding block diagram and Fig. 2 a schematic view of the 3D-ISM.

The aim of given investigation is to study the effect of cooling upon rat hepatocyte structure using transmission electron microscopic and computer morphometric methods. Ultrastructural and morphometrical characteristics of hepatocytes under liver cooling for various levels under in vivo and in vitro conditions were investigated. Vistar rats of 180-250 g were used in the experiment. Liver cooling (in vivo) was performed by means of original cryoapplicator with different probe temperature (1,2). Liver tissue for transmission electron microscopy was fixed in glutaraldehyde fixator on cocadylate buffer and OsO4. Dehydration was completed on acetone (3). Tissue embedding was done into the mixture of Epon/Araldite epoxy rasin. Ultrathin slices were contrasted by the method of Reinolds. Cell viewing and imaging were accomplished by electron microscope at accelerating power of 75kV.

Morphometrical and stereometrical analysis was performed using the “Morpho-Tools” original computer system (c) 1994-1996 A.S. Kaprelyants, A.A. Kaprelyants, A.N. Reylan .

Extended junctional sarcoplasmic reticulum (EJSR) is an invariant differentiation of the sarcoplasmic reticulum (SR) in bird cardiac myocytes (CM) and central to excitation-contraction coupling (ECC). EJSR occurs as both continuous and discontinuous extensions of junctional sarcoplasmic reticulum (JSR), and surrounds and pervades the Z/I band as the “ EJSR Z-rete” whose geometry has mechanistic implications for the function of “couplings” in ECC, in general. “Peripheral coupling(s)” (PC) in birds, and the additional “interior coupling(s)” (IC) at transverse tubules (TT) in mammals, are formed by tight apposition to plasmalemma of JSR, a specialized calcium (Ca) store of the SR. Free SR (FSR; i.e. free of JSR/EJSR specializations) is the rest of the smooth, tubular SR network, which connects intercalated patches of EJSR forming the EJSR Z-retes and, elsewhere, displays both longitudinal and transverse geometries in surrounding the contractile material for the purpose of sequestering Ca after each muscle contraction. Except for EJSR having no plasmalemmal contact, morphologically, EJSR and JSR are homologues:1 both have similar sizes; are studded (approx. 32 nm center-to-center) with junctional processes (JP; ryanodine receptor (RyR)/-Ca-release channels);

“Coupling(s)” (Fig. 1) are structure complexes in cardiac muscle consisting of junctional SR (JSR) tightly apposed to the cytoplasmic side of the plasmalemma. They are either peripheral (PC; when on the cell surface) or interior (IC; when on transverse tubule(s), TT). The JSR is a store for calcium (Ca)-release through Ca channels/ryanodine receptors in junctional processe(s) (JP) on the JSR surfaces. Action potential(s) (AP) for excitation-contraction coupling translate, as mediated by L-type channels, into Ca-entry into a ˜ 20 nm junctional gap (JG) containing JP between plasmalemma and the JSR's junctional membrane proper. In mammalian cardiac myocyte(s) (CM), where IC are ubiquitously distributed along TT at Z-bands, an AP quickly effects almost simultaneous global contraction, even in the center of cells of large diameter, whence CICR can be viewed as a one-step event between Ca-entry and JSR Ca-release from couplings. Avian CM, however, have no TT and, thus, only PC.

We have previously reported that epithelial cells appear to be clustered in the perilobular stroma of the human lactating breast', that phenomenone has not beendocumented well in the literature. The present study is aimed at to clarify the nature and characteristics of the clustered-epithelial cells in relation to basement membrane, epithelial cell death and myoepithelial cells.

We studied the mammary gland of Wister female rats in various stages of resting, pregnant and lactating phase histochemically and electron microscopically. Basement membrane was detected by immunohistochemically (indirect peroxidase method) on 4% paraformaldehyde (PFA)-fixed frozen section using anti type rv collagen (goat, Southern Biotechnology Assoc. Inc. Birmingham, AL, USA) and electron microscopcally. Apoptotic cell death was shown by in situend labeling method on formalin-fixed paraffin-embedded section. The myoepithelial cells were observed by alkaline phophatase Azo dye method on frozen section, and by immunohistochemical staining for actin (smooth muscle) (clone; 1A4, Sigma Chemical Co. St Louis, MO, USA).

Previously, we reported the presence of caveolae, clathrin-coated pits and vesicles in toad urinary bladder granular epithelial cells, and in smooth muscle and endothelial cells. Since then we investigated the occurrence of caveolae in the rabbit urinary bladder cells. Here we report for the first time the presence of caveolae and clathrin-coated pits and vesicles in the granular epithelial, smooth muscle and endothelial cells of rabbit urinary bladders.

Urinary bladder sacs were surgically removed from doubly pithed toads and anaesthetized rabbits, and allowed fixation for 1 hr in 2% glutaraldehyde in PIPES buffer. These bladder tissues were divided for scanning (SEM) and transmission electron microscopy (TEM) for comparative analysis. A postfixation using 1% osmium tetroxide was carried out for 1 hr. Tissues were appropriately processed for SEM and TEM observations. Ultrathin sections, made with a diamond knife, were taken on bare nickel grids and then exposed to uranyl acetate and lead citrate for viewing in the TEM.

Interstitial cells of Cajal (ICCs) were first described as primitive neurons found within organs innervated by autonomie nerves. In the gut, ICCs are juxtaposed among enteric nerve fibers and smooth muscle cells, suggesting they may modulate enteric neurotransmission and affect motility. The recent discovery of neurokinin-1 receptor (NKrl)-like immunoreactivity (ir) on ICCs has strengthened this hypothesis. This study compared the distribution of NKlr-ir to the staining patterns of other reported markers of ICCs including cholera toxin subunit b (CTB), neuron specific enolase (NSE), NADH diaphorase, NADPH diaphorase, nitric oxide synthase (NOS), and vimentin. Albino male rats were anesthetized and whole mount preparations of myenteric plexus-longitudinal muscle with attached circular muscle were dissected. ICCs were stained using multi-label techniques and cells were visualized with a Sarastro 2000 confocal laser scanning microscope equipped with Image Space software.

In the small intestine, ICCs associated with circular muscle, but not longitudinal muscle, expressed NKlr-ir.

Microcysts are most evident in the posteroventral and anteroventral cochlear nucleus (PVCN & AVCN) of the gerbil. They are reported as a neurodegenerative disorder or spongioform degeneration or a dynamic process related to the degree of auditory stimulation in the cochlear nucleus of the gerbil. Little information is avalable on the origin and formation of microcysts in the cochlear nucleus of the gerbil. The aim of this study was to investigate the internal morphologic changes of microcysts in the gerbil PVCN during postnatal development by scanning electron microscopy.

The mongolian gerbil, Meriones unguiculatus, were sacrificed by cardiac perfusion of a saline nitrite flush, followed by a 3% glutaraldehyde in 0.1M phosphate buffer. After fixation the brain were washed in 0.1M phosphate buffer ,embedded in 5% agar, vibratome-sliced through the cochlear nucleus, and then postfixed in the 1% osmium tetroxide in 0.1M phosphate buffer for one hour. The sectioned tissues were dehydrated in a graded ethanol series to absolute ethanol and transferred to liquid carbon dioxide for critical point drying.

It has been considered that the behavior of tumors is consistent with the concept that tumor growth is angiogenesis dependent. However, additionally to the newly formed vessels other patterns of tumor capillaries have been described. Information concerning the ultrastructure of tumor blood vessels has been obtained mostly from experimental models. With regard to human tumors, since no systematic ultrastructural studies are available on the gastric tumor microvasculature, the aim of the present work was to characterize the vascular architecture of this type of tumor.

Surgically resected specimens were obtained in tumors from five patients (three males and two females) between 45 to 66 years of age (mean, 56.2 years), with a histopathological diagnosis of moderately differentiated gastric adenocarcinoma. Immediately after resection, tissue samples taken from areas of tumor center were processed by routine transmission electron microscopy techniques and observed in a Hitachi H-500 EM.

The microvasculature within gastric adenocarcinomas was characterized by newly formed capillaries with proliferative endothelial cytoplasm (Fig. 1).

Prominent cytoplasmic vacuoles in renal tubular epithelial cells of the outer medulla were observed in several kidneys from test article-dosed mice during routine light microscopic (LM) examination. Because the vacuolar change was seen infrequently by LM examination, and was not found in any control mice from that study, it was not clear whether the vacuolation represented a drug-induced change. To address this question, kidney sections from multiple unrelated mouse studies (214 control mice and 541 test article-dosed mice representing six different compounds) were examined by LM for similar vacuolar changes. Vacuolation was seen by LM in 2.3% of the control and 2.8% of the test article-dosed mice. Transmission electron microscopy (TEM) was performed on kidneys with prominent vacuoles seen by LM in 5 control mice and 2 test article-dosed mice to further characterize the vacuoles. Additionally, kidneys from 4 randomly selected control mice lacking vacuolation by LM were examined by TEM. Samples from the outer medulla of kidney that had been fixed by immersion in 10% formalin were post-fixed in 2.5% glutaraldehyde and 2% osmium tetroxide and processed routinely for TEM.

The purpose of this study was to compare the biological response to two bone substitute materials, hydroxyapatite cement (HAC) and demineralized bone (DMB), when placed in canine cranial bone. The implants were surgically positioned in 15 mm diameter defects created in the parietal plate, harvested en bloc at 3 months (m) and 6m postop, and fixed in 70% ethyl alcohol to preserve the xylenol orange bone label. Half of each implant site was processed into paraffin and the other half into Spurr plastic resin. In order to evaluate the osseoinductive properties of DMB, implants were also surgically placed in the rectus femorous muscle, harvested en bloc at 3m post op, fixed in 0.1 M Na phosphate buffer, pH 7.2, decalcified in 5% EDTA, pH 7.2, and embedded in plastic. All implants were evaluated with light microscopy (LM), radiography, energy-dispersive spectroscopy (EDS), and transmission electron microscopy (TEM). Tissue for LM was routinely decalcified with SFFA, embedded in paraffin, sectioned, stained with hematoxylin/eosin, and viewed with a Zeiss transmitted-light photomicroscope [Figs. 1, 2, 5].

Atherosclerosis is a degenerative arterial disease that leads to the gradual blockage of vessels due to plaque formation or acute ischaemic events such as plaque rupture. A thorough understanding of plaque morphology is necessary in the determination of factors underlying coronary artery disease. Intravascular ultrasound (IVUS) represents a diagnostic technique that provides tomographic visualization of coronary arteries. Ultrasound reflects sound at the interfaces between media of different acoustic refractive indices theoretically implying that various components within an ultrasound image should be distinguishable. The aim of this study is to classify plaque lesions using advanced digital image processing into the following categories: adventitia, media, fibrous, necrotic core, and calcified. Examination of plaque composition can yield valuable information necessary in determining the appropriate preventative and mechanical interventions.

Diseased samples were obtained from excised human coronary arteries at autopsy. Intravascular ultrasound images were acquired using an HP SONOS clinical IVUS imaging system and 3.5 French 30 MHz catheters.

Morphologic changes in Alzheimer’s disease (AD) include a decrease in synapse number while the surviving synapses are increased in size by a process known as compensatory synaptogenesis. There is disagreement among investigators as to whether the synapse changes observed in AD in humans also occurs in the aging rat. The hippocampus, plays a significant role in learning and memory in both humans and rats and is known to be particularly susceptible to cell loss associated with aging. s In the rat hippocampus, age-related synaptic density declines as much as 27% have been reported by some authors, while other investigators have failed to detect a change in synapse number with age. A secondary factor in the total number of synapses in the dentate gyrus is the number of granule cells. Proliferation or regeneration (neurogenesis) of hippocampal granule cells is known to occur well into the adult life of the rat. The goal of the current study was to further explore age-related changes in rat hippocampal synapses and granule cells using the disector morphometry method.

During hematopoiesis of mammalian animals, an erythroblastic island in the bone marrow has been reported to contain a central macrophage surrounded by many erythroblasts. Fetal mouse livers and spleens, which are treated with collagenase, have been used to get the erythroblastic island in tissue cultures. In this study, we have cultured some stamped cells obtained from spleens of anemic mice and examined their structures by scanning electron microscopy.

Hemolytic anemia of female BALB/c mice was induced by acetylphenylhydrazine (4mg/g body weight) injection for 5 days. Spleens were resected on the second day after the last injection and a “spleen-stamp culture“ method was performed. Briefly, they were cut with razor blades (Fig. 1a) and splenic cells on the cut tissue surface were stamped on collagen-coated coverslips (Fig.1b). The stamped splenic cells were covered with dialysis membranes (Fig. 1c). They were cultured with 55% IMDM, 20% L-15 and 25% FBS, containing 2U/ml erythropoietin (EPO) in humidified atmosphere of 5% CO2 at 37°C for 1-3 days.

The Biological Microscopy and Image Reconstruction Resource (BMIRR) is operated by the Wadsworth Center as a national biotechnology resource, with funding through the NIH Center for Research Resources and from NSF. This biological imaging resource has evolved continuously over the past two decades. Early development focussed on correlative, same-cell light and electron microscopic techniques, combining the capabilities of video-enhanced light microscopy and high-voltage electron microscopy. A current area of development is electron microscopic tomography, whereby the full 3D capabilities of higher voltage (400-1200 KV) electron microscopy is brought to bear on biological problems. In particular, the recent development of techniques for merging projection data from two mutually perpendicular tilt series has permitted significantly improved resolution, reducing the missing wedge of information to a missing pyramid. Attention is now turning to optimization of conditions for applying tomography to frozen-hydrated specimens, using automated data collection on our cryo-IVEM. Combined with parallel advances in same-cell manipulation and viewing, the BMIRR provides biologists with a unique combination of imaging and computational tools for research into the 3D structure and dynamics that underly cellular processes (see figures and refs. 2-4).

We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.

Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.

We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.

The intermediate high-voltage electron microscope (IVEM) located at the National Center for Microscopy and Imaging Research at San Diego (NCMIR) can image relatively thick specimens that contain substantial three-dimensional (3-D) structure. Electron tomography is an important tool used at NCMIR for deriving 3-D cellular and subcellular structure from IVEM images. Reconstruction algorithms commonly used in electron tomography include weighted back projection, and iterative algebraic reconstruction techniques such as ART and SIRT. Improvements in reconstruction quality are possible using the iterative algorithms. Because these algorithms are computationally intensive, we have ported them to massively parallel computers at the San Diego Supercomputer Center, reducing the computation time over that required with workstation level machines.

The quality of tomographic data for the 3-D reconstruction of biological structures is also being enhanced by NCMIR projects to improve the microscope. We have designed and constructed special electron optics and microscope control systems for the JEOL 4000EX.

The STEM facility at Brookhaven National Laboratory has been in operation since Oct. ‘77, using a custom-built instrument (STEM1) with cold field emission source, 2.5Å probe, -150°C cold stage, efficient dark field detectors and computer control & data acquisition system. A specimen changing air lock and several portable vacuum chambers permit vacuum transfer of specimens from a separate vacuum system where they were freeze dried overnight.

The large angle dark-field signal produced by the STEM is directly proportional to the total mass within the probed area. STEM mass mapping is based on this linear relationship and the fact that only specimen-specific atoms remain on the substrate after washing with volatile buffer and freeze drying. All images are digital and available via Internet. PC software can be provided for analysis.

STEM mass accuracy ranges from a fraction of a percent on well-defined individual particles such as viruses in the 50 MDa to 10 GDa range, to ∼1% around 1 MDa and ∼10% in the 50 kDa range.

The National Center for Macromolecular Imaging (NCMI) is a resource supported by the National Center for Research Resources (NCRR) of NIH. Its mission is to advance electron imaging of macromolecular assemblies to near atomic resolution. We have focused on biological assemblies which are too large or too complex to be studied by conventional x-ray crystallography or NMR spectroscopy techniques. As is the case with all the NCRR-supported facilities, we also conduct methodology development, collaboration, service, technology dissemination and training.

The objectives of this Research Resource are to develop the technology of high resolution electron cryomicroscopy of biological macromolecules, assemblies and crystals in an integrated approach that includes specimen preparation, hardware and software development. A current methodology development includes the characterization of the 1k x 1k slow-scan CCD camera for both electron diffraction and imaging of ice-embedded specimens. These studies have defined the advantages and limitations of the CCD camera for electron crystallographic analysis. Another core research project deals with the problems of specimen movement due to the electron beam and/or charging and seeks to minimize these phenomena. Our efforts have resulted in a better understanding of the physics of charging. We have modified a Gatan ion-beam coater so that we can apply a thin layer of carbon onto ice-embedded specimens.