High-resolution Light Microscopy Research Articles

Frontal filament morphogenesis in the salmon louse Lepeophtheirus salmonis.

The objective of the present study was to understand how and when the frontal filament (FF) in the salmon louse Lepeophtheirus salmonis is produced by examining the sequence of morphological changes leading to FF production in the copepodid and early chalimus stages. Atlantic salmon (Salmo salar) were heavily infested with newly molted copepodids. Sea lice were sampled prior to infestation and at 1, 2, 3, 4, 5, 6, 7, 8, and 9 days postinfestation. FF morphogenesis from newly molted copepodid to chalimus II, i.e., through 2 molts, was studied using high-resolution light microscopy of serial transverse and sagittal resin sections. Three groups of cells, identified as A, B, and C, are thought to be involved in the production of the secretions (S1 and S2) that form the filament material. The amount and shape of S1 and S2 and their association with B- and C-group cells, respectively, changed with the molt cycle. The following scenario for FF development is proposed: the first secretion to form after the molt for both copepodid and chalimus stages is S1, and it is formed by B-group cells and becomes the basal plate of the external FE C-group cells produce S2 during mid-intermolt to premolt stage. The S2 becomes the stem of the external FE In premolt larvae, S1 and S2 were contained within a cuticle-lined invagination that had a form similar to that of the extruded filament. The axial duct present in both copepodid and chalimus originates from the A-group cells and probably carries a secretion used to attach the filament to the host. This study provides strong evidence that L. salmonis produces a new filament with each molt, creating the possibility of using a sea lice control method based on interference with filament production more feasible.

Chondrogenesis of the branchial skeleton in embryonic sea lamprey, Petromyzon marinus.

This study provides concise temporal and spatial characteristics of branchial chondrogenesis in embryonic sea lamprey, Petromyzon marinus, using high resolution light microscopy, transmission electron, and immunoelectron microscopy. Prechondrogenic condensations representing the first branchial arch appeared first in the mid-region of the third pharyngeal arch at 13 days post-fertilization (pf). Cartilage differentiation, defined by the presence of the unique, fibrillar, non-collagenous matrix protein characteristic of branchial cartilage, was first observed at 14 days pf. Development of lamprey branchial cartilage appeared unusual compared to that in jawed fishes, in that precartilage condensations appear as a one-cell wide orderly stack of flattened cells that extend by the addition of one dorsal and one ventral condensation. Development of lamprey gill arches from three condensations that fuse to form a single skeletal element differs from the developing gill arches of jawed fishes, where more than one skeletal element forms from a single condensation. The initial orderly arrangement of cells in the lamprey branchial prechondrogenic condensations remains throughout development. Once chondrification of the condensations begins, the branchial arches start to grow. Initially, growth occurs as a result of matrix secretion and cell migration. Later in development, the arches grow mainly by cell proliferation and enlargement. This study defines the morphology and timing of lamprey branchial chondrogenesis. Studies of lamprey chondrogenesis provide not only insight into the developmental biology of a unique non-collagenous cartilage in a primitive vertebrate but also into the general evolution of the skeletal system in vertebrates.

The dynamics of acentric chromosomes in cancer cells revealed by GFP-based chromosome labeling strategies.

Autonomous replicons, such as viral episomes and oncogene containing double minute chromosomes (DMs), lack centromeres and consequently should be lost rapidly when the nuclear membrane breaks down at mitosis. Surprisingly, they are not. This raises the important question of the mechanisms that enable their efficient transmission to daughter cells. We review recent developments in GFP-based chromosome labeling strategies that enable real time analyses using high resolution light microscopy to provide insights into this issue. The results reveal that episomes and DMs both adhere to host chromosomes, a process referred to as "chromosome tethering". Such association enables acentric molecules to use the chromosomal centromere in trans, thereby achieving efficient transmission to daughter cells. This unique mechanism of mitotic segregation also raises the possibility of developing a new class of anti-cancer drugs that work by selectively eliminating growth enhancing genes from cancer cells. J. Cell. Biochem. Suppl. 35:107-114, 2000.

Functional reconnection of severed mammalian spinal cord axons with polyethylene glycol.

We describe a technique using the water-soluble polymer polyethylene glycol (PEG) to reconnect the two segments of completely transected mammalian spinal axons within minutes. This was accomplished by fusing completely severed strips of isolated guinea pig thoracic white matter maintained in vitro in a double sucrose gap recording chamber. The faces of the severed segments were pressed together, and PEG (MW 1,400-3,500 d; approximately 50% by weight in distilled water) was applied directly to this region through a micropipette and removed by aspiration within 2 min. Successful fusion was documented by the immediate restored conduction of compound action potentials through the original transection and by the variable numbers of fused axons in which anatomical continuity was shown to be restored by high-resolution light microscopy and by the diffusion of intracellular fluorescent dyes through fused axons. These data support the conclusion that some severed and subsequently PEG-fused spinal axons both demonstrate restored anatomical continuity and also are physiologically competent to conduct action potentials. This work adds to our previous demonstration that PEG application can immediately repair severely crushed, rather than cut, spinal cord white matter, and may lead to novel treatments for acute trauma to the central and peripheral nervous systems.

Early events in naphthalene-induced acute Clara cell toxicity: comparison of membrane permeability and ultrastructure.

Naphthalene causes severe dose- and site-selective injury to mouse nonciliated bronchiolar (Clara) epithelial cells. Toxicity is characterized by exfoliation of injured Clara cells into the airway lumen 24 h after exposure. The purpose of this study was to define the temporal pattern of intracellular changes immediately following naphthalene treatment, with the goal of identifying critical early events involved in cytotoxicity. Mice were injected with naphthalene or carrier and were killed 1, 2, 3, and 6 h after treatment (PT). Loss of membrane integrity was assessed by ethidium homodimer-1 permeability and confocal microscopy. Cell morphology and ultrastructure were evaluated using high-resolution light and electron microscopy. Permeable cells were found only in terminal bronchioles and increased in abundance with time PT. At 2 and 3 h PT, when most Clara cells had early signs of injury, few permeable cells were detected. Many Clara cells had apical membrane blebs that contained abundant, swollen, smooth endoplasmic reticulum (SER) and few other organelles. By 6 h PT many Clara cells were membrane-permeable. However, many permeable Clara cells lacked apical blebs and SER was less abundant in these cells. Cytoplasmic blebbing may be a mechanism to protect the cell by isolating and removing damaged SER. We conclude that the early stages of injury include SER swelling and bleb formation which precede increases in cell membrane permeability after acute naphthalene injury to bronchiolar Clara cells in vivo.

Postnatal development of perisomatic GABAergic axon terminals on neurons projecting from area 17 to area 18 of the cat visual cortex.

We have studied the postnatal development of presumptive axon terminals (puncta) which were recognized by antibodies to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and were located on the somata of area 17 neurons projecting to the ipsilateral area 18 of the visual cortex in cats ranging from 7 days of age to adulthood. Projection neurons were retrogradely labeled by injection of horseradish peroxidase conjugated to wheat germ agglutinin into the ipsilateral area 18. These neurons were mainly pyramidal in shape at all the developmental stages examined and the adult distribution of labeled cells was reached by 21 days. Subsequent GABA postembedding immunohistochemistry using high-resolution light microscopy was carried out to study the development of GABAergic terminals on cell bodies of identified projecting neurons in layers II-III. At all ages examined, we found perisomatic GABAergic puncta on these cells. Their density showed a significant increase from postnatal days 7 to 45, and then remained largely constant through adulthood. Since GABAergic puncta are considered the light-microscopic correlate of GABAergic synaptic terminals, our results support the idea of a developmentally regulated increase in the inhibitory activity of local interneurons on area 17 pyramidal neurons projecting to area 18 in the cat visual cortex which occurs within the same time frame as that of the acquisition of the mature operation of these cells.

Homologous association of the Bithorax-Complex during embryogenesis: consequences for transvection in Drosophila melanogaster.

Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans, presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how early trans-sensing occurs nor the extent or duration of the underlying physical interactions. We have investigated the physical proximity of homologous genes of the BX-C during Drosophila melanogaster embryogenesis by applying fluorescent in situ hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos, and is not perturbed by the transcriptional state of the genes throughout embryogenesis. Pairing frequencies never reach 100%, indicating that the homologous associations are in equilibrium with a dissociated state. We determined the effects of translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, on the extent of diploid homologue pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm of chromosome 3 or to the X chromosome abolished trans-regulation of the Ultrabithorax gene, pairing of homologous alleles surprisingly was reduced only to 20-30%. A zeste protein null mutation neither delayed the onset of pairing nor led to unpairing of the homologous alleles. These data are discussed in the light of different models for trans-regulation. We examined the onset of pairing of the chromosome 4 as well as of loci near the centromere of chromosome 3 and near the telomere of 3R in order to test models for the mechanism of homologue pairing.

Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)

Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)

Pollen Tube Distribution in the Kiwifruit (Actinidia deliciosaA. Chev. C. F. Liang) Pistil in Relation to its Reproductive Process

High resolution light and fluorescence microscopy were used to investigate the structural and cytochemical features of the kiwifruit(Actinidia deliciosa)pistil and to follow the pollen tube pathway after pollination. The multicarpellary ovary is syncarpous only at the ovary level thus leaving 30–40 free styles on top. The fusion of the longitudinally-folded carpels to form the syncarpous ovary forms a central parenchymatous axis, the columella, from which the ovules radiate outwards into the ovary cavity. A prominent cup shaped depression on the columella at the top end of the ovary, termed the pollen tube distributor cup (PTDC) was detected. Pollen tubes from the stigma travel through the transmitting tract and enter the PTDC from where they are distributed towards the ovary. Even when pollination is restricted to two stigmas, the PTDC seems to ensure that the pollen tubes are evenly distributed around the ovary resulting in an even distribution of seeds. This suggested role of the PTDC which could compensate for over and under pollination of individual stigmatic arms is another adaptive feature which plays a crucial role in the reproductive process of kiwifruit. The significance of this structure for pollination by insects is discussed.

Bioactive surface coatings for nanoscale instruments: effects on CNS neurons.

A method is described for depositing onto medical instruments highly biocompatible and bioactive surface coatings that can promote and stabilize cell attachment. The coatings were made by first depositing thin films of materials, such as diamond-like carbon, or metals, including tantalum, tungsten, platinum, gold, iridium, palladium, and brass. These surfaces were further altered to either promote or inhibit cell growth and spreading by an additional overcoat of biological materials, including the extracellular matrix proteins, laminin, fibronectin, and collagen IV. The deposition technique used a metal or carbon plasma, and the important properties of film adhesion, hardness, density, and smoothness are tailored by control of the ion bombardment energy. The films are translucent enough to permit high resolution light microscopy for rapid and detailed examination of tissue response. These bioactive substrates have been tested on primary central nervous system neurons, and the growth response is excellent. Equally successful have been our attempts to anchor neurons, without associated proliferation of non-neuronal cells, using coatings of poly-d-lysine. The method and the materials could have important ramifications in a number of areas of research and biotechnology, for example for chronic implantation of microelectrode arrays in the cerebral cortex for neuroprosthetic and neural monitoring application and for research on the human central nervous system. Possible application in nonneuronal fields, such as for coronary artery stents and pacemaker electrodes, also are discussed.

Monitoring of cellular signalling and metabolism with modular sensor-technique: The PhysioControl-Microsystem (PCM ®)

Microsensors provide instruments particularly suited for the noninvasive analysis of cell and tissue cultures. The outstanding benefit lies in the passive behaviour of continuously working transducers, which in turn allows the dynamic recording of function-specific cellular processes. The microsensor system presented in this paper is a modular arrangement of various planar and non-planar sensor elements surrounding small cell culture chambers. An optic access to the cultures (e.g. for high resolution light microscopy and spectro-photometric techniques) enables a parallel and comparative data acquisition. The system was originally designed for biomedical research in chemotherapy and pharmacology but it proved to be an effective device both for toxicological and environmental research.

Chapter 13 Perfusion Chambers for High-Resolution Video Light Microscopic Studies of Vertebrate Cell Monolayers: Some Considerations and a Design

Publisher Summary This chapter defines a perfusion chamber as a vessel, mounted on the stage of a light microscope that enables a living specimen to be followed continuously by high-resolution light microscopy (LM) while also allowing the fluid bathing the specimen to be rapidly exchanged during the observation period. Perfusion chambers are employed in a variety of biological and biomedical studies of cells and tissues. As they allow the media bathing the specimen to be removed and replenished, they are widely used to maintain specimen viability during long-term investigations, to study the effects of environmental changes, and to periodically sample for the production of cellular products. Perfusion chambers are also employed to deliver tracers for uptake studies and to assay the effects of drugs and other biologically active components on cell/tissue structure and function. The chapter discusses the topic in two-fold manner: (1) the first is to identify some of the variables that must be considered when constructing or choosing a perfusion chamber for high-resolution video-enhanced LM studies. (2) The second is to describe a rugged and simple laminar-flow chamber used in our laboratory for short-term (12-day) studies of vertebrate cell monolayers.

Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: An overview

Several microscopical techniques, such as high resolution light microscopy, Normaski microscopy, laser confocal and transmission electron microscopy, were used in a correlative morphological study of human osteoarthritic (OA) cartilage. Emphasis was made on the characterization of chondrocytes heterogeneity observed in this tissue. Novel findings were assessed in the morphological and immunocytological study of the chondrocytes organized in aggregates or “clones” typical of this degenerative disease, consisting of the modification of certain elements of the cytoskeleton that influence changes in the cell shape. Also, the presence of cilia and centrioles found in certain cells raised the question if chondrocytes are able to move and regroup as an alternative mechanism to mitosis in the formation of cell clusters or “clones.” The presence of two types of secretory chondrocytes was observed and discussed. The use of a correlative approach of several microscopical techniques in a systematic morphological and immunocytological characterization of chondrocyte population within the fibrillated and nonfibrillated human osteoarthritic cartilage gave complementary information that could be important for a better understanding of the histopathogenesis of OA. Microsc. Res. Tech. 40:22–36, 1998. © 1998 Wiley-Liss, Inc.

Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: an overview.

Several microscopical techniques, such as high resolution light microscopy, Normaski microscopy, laser confocal and transmission electron microscopy, were used in a correlative morphological study of human osteoarthritic (OA) cartilage. Emphasis was made on the characterization of chondrocytes heterogeneity observed in this tissue. Novel findings were assessed in the morphological and immunocytological study of the chondrocytes organized in aggregates or "clones" typical of this degenerative disease, consisting of the modification of certain elements of the cytoskeleton that influence changes in the cell shape. Also, the presence of cilia and centrioles found in certain cell raised the question if chondrocytes are able to move and regroup as an alternative mechanism to mitosis in the formation of cell clusters or "clones." The presence of two types of secretory chondrocytes was observed and discussed. The use of a correlative approach of several microscopical techniques in a systematic morphological and immunocytological characterization of chondrocyte population within the fibrillated and nonfibrillated human osteoarthritic cartilage gave complementary information that could be important for a better understanding of the histopathogenesis of OA.

The mechanism of positive scintigraphy with 99mTc-pertechnetate in adenolymphomas of the parotid grand

In order to clarify the mechanism of positive scintigraphy with Tc pertechnetate in adenolymphomas of the parotid gland, 10 cases of adenolymphoma were studied morphologically and from a functional view point. Salivary tissue obtained from normal parotid glands were also investigated for comparison with the tumor. In morphological studies, rapid tissue fixation by microwave irradiation produced very good results for examining mitochondria by high resolution light microscopy. A number of mitochondria were found both in the epithelial cells of adenolymphoma and in the striated duct cells of the parotid gland. In functional studies, the one-step method of enzymatic histochemistry was used for detecting Na-K ATPase. Na-K ATPase was found on the baso-lateral side of the epithelial cells of adenolymphoma and the striated duct cells of the parotid gland, and this activity of Na-K ATPase may enhance the accumulation of Tc pertechnetate in these cells. These observations may provide evidence for the bi-cellular theory which indicates that adenolymphoma originated from immature intercalated duct reserve cells.

Vascular tissue adaptations in end-to-end autologous arterial grafts in rats: a morphometric analysis.

Autologous vein grafts are employed extensively to bypass stenoses in the arterial circulation. More recently arterial segments have been used for such bypass surgery. In this study the adaptation of regenerating vascular tissues in experimental autologous artery grafts (4 mm long and 1 mm in diameter) in 20 adult male Wistar rats was analysed. At 1, 2, 4, 8 and 16 wk after insertion, 4 grafts per time interval were removed, processed for high resolution light microscopy and the thicknesses of the media and neointima, as well as the area fractions of smooth muscle cells, were analysed morphometrically. All grafts were reendothelialised by 2 wk. Neointimal hyperplasia (a subendothelial layer of smooth muscle cells) developed in all grafts and reached its maximal thickness (40.4 +/- 4.7 microns) at 2 wk. The area fraction of smooth muscle cells in the neointima of the artery grafts did not change significantly at any time from 2 to 16 wk. The media underlying the neointima of the artery grafts remained relatively constant throughout the 16 wk duration of the experiment. Whilst the total wall thickness of the grafts reduced significantly between 2 and 4 wk after insertion, at all times the grafts were thicker than the host artery.

Apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium of the adult rat.

It is widely assumed that oestrogen administration in the male mimics hypophysectomy by suppressing gonadotrophin secretion. Nevertheless, oestradiol treatment can increase germ-cell apoptosis mainly at stages IV-X of the spermatogenic cycle, rather than at stage VII when apoptotic germ-cell death is mainly triggered by gonadotrophin withdrawal caused by hypophysectomy. Since the roles of testicular oestrogens in spermatogenic regulation, if any, are unknown, we re-evaluated the germ-cell types that undergo apoptosis after oestradiol treatment. Adult male rats were injected daily with 50 micrograms oestradiol, oestradiol plus testosterone propionate (25 mg every 3 days) or oestradiol plus human menopausal gonadotrophin (equivalent to 25 iu FSH plus 25 iu LH) for 15 days. Apoptosis was assessed by in situ 3'-end labelling of internucleosomal DNA fragments in plastic semithin sections; the germ-cell types involved were identified by high-resolution light microscopy. The quantitative analysis of our results shows that the apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium differs from that reported to be caused by gonadotrophin or testosterone withdrawal, suggesting a possible role for oestradiol in the modulation of germ-cell death in the adult testis of the rat.

Improved High Resolution Light Microscopy and Transmission Electron Microscopy Techniques in Diagnostic Pathology

There is little argument concerning the usefulness of high resolution light microscopy (HRLM) and transmission electron microscopy (TEM) in diagnostic pathology. However, the sophisticated histotechnology, developed for ultrastructural research, can be adapted to cope with present demands of modern surgical pathology. In our laboratories we have introduced changes in handling the tissues to avoid blind, limited and fragmented samples. The use of fast penetrating, environment-resistant fixative has allowed larger samples. Embedding large biopsies in versatile epoxy Spurr has produced generous thick and thin sections, avoiding waste of diagnostic material and resulting in more accurate diagnosis. Better contrast of the sections was obtained by staining with basic fuchsin and toluidine blue. Coverslipping has prevented scratches and dirt on the sections. These adaptations of the methods for HRLM-TEM allow better utilization of light and electron microscopes for modern surgical pathology. (The J Histotechnol 20:45, 1997)

Immunocytochemical Demonstration of Hormones in Pancreatic and Pituitary Tissue Embedded in Methyl Methacrylate

We describe the immunocytochemical demonstration of hormones in pancreatic and pituitary tissue embedded in the plastic methyl methacrylate. Semithin sections were prepared for high resolution light microscopy and a broad spectrum of antibodies applied. Excellent immunocytochemical staining was reliably achieved with all the antibodies investigated combining strong positivity with good tissue morphology. The results were superior to similar studies we have previously performed with other acrylic plastic embedding media, and the method can be routinely employed in a diagnostic laboratory. (The J Histotechnol 20:35, 1997)