Scanning tunneling microscopy of human erythrocyte membranes.

Images of surfaces of human erythrocyte ghosts, lecithin liposomes, spectrin, erythrocyte membrane skeleton, concanavalin A and concanavalin A--decorated erythrocyte ghosts were obtained by scanning tunneling microscopy. The dimensions and surface topography of some membrane structures are described and discussed.     

Scanning tunneling microscopy with applications to biological surfaces

Each major advance in the field of microscopy has eventually been translated into major advances in the biological and medical sciences. The scanning tunneling microscope (STM) offers exciting new ways of imaging biological surfaces with resolution to the sub-molecular scale. Rigid, conductive surfaces can readily be imaged with the STM with atomic resolution. Unfortunately, few biological surfaces are sufficiently conductive or rigid enough to be examined directly with the STM. At present, non-conductive surfaces can be examined in two ways: 1) Sufficiently thin molecular layers attached to conductive substrates so that tunneling can occur through the molecules; or 2) coating or replicating non-conductive surfaces with metal layers so as to make them conductive, then imaging with the STM. We present images of biological and organic molecules obtained with these techniques that demonstrate the possibilities and limitations of each. Future advances leading to atomic resolution STM of biological surfaces depend on significant progress in the art and science of making biomaterials compatible with the restrictions of the instrument.     

Scanning electron microscopy of attached trophozoites of pathogenic Entamoeba histolytica

The surface morphology of Entamoeba histolytica trophozoites of HM 1:IMSS (axenic and monoxenic) and HK9 (axenic) strains cultured on plastic and MDCK cell substrates was examined using scanning electron microscopy (SEM). The conditions for processing trophozoites were determined by comparing the SEM observations with the morphology of living amebas examined by light microscopy. The most frequent surface differentiations in all the amebas observed with SEM were lobopodia. Round cytoplasmic projections were found in approximately half of the axenic amebas. Endocytic stomas and filopodia were more common in monoxenic cultures while the uroid was found in only 2-8% of all examined amebas. The basal surfaces of the trophozoites, involved in both attachment and cytolysis, showed no unusual features, except for the presence of a small number of short filopodia at the outer edge. No differences were found in the morphology of amebas grown on artificial and natural substrates. These observations demonstrate that there are significant quantitative differences in the surface morphology of cultured trophozoites of different strains of E. histolytica and that association with bacteria produces an increase in the relative number of surface specializations of the parasite.     

Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells.

This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as a visualized by scanning electron microscopy. Treponemes were incubated for 3 hr with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without altering the morphological integrity of the cultured cells. Attachment appeared to involve a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum.     

Scanning electron microscopy of cell surface antigens labeled with colloidal gold

A method is described and discussed that permits the specific labeling of the surface of prefixed cells with the colloidal gold marker viewed with the scanning electron microscope. Its value depends exclusively on the use of backscattered electron imaging. Its advantages include the possibility of preserving the surface features of the labeled cells, the ease with which specificity can be established, the possibility of making total counts of the labeled surface antigenic sites, and the possibility of achieving distinct labeling for two different antigens expressed on the surface of the same cell.     

Scanning tunneling spectroscopy investigation of self-assembled plastocyanin mutants onto gold substrates under controlled environment

The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/-1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed.     

Scanning tunnelling microscopy in biotechnology.

The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment.     

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.     

Application of scanning tunneling microscopy to structural biology

Scanning tunneling microscopy offers the possibility of visualizing biological molecules in conditions similar to those in vivo with molecular resolution. Images of DNA and various proteins have been obtained, but insufficient conductivity through, and inhomogeneous and unstable adsorption of the biomolecules continue to prevent reliable imaging. Applying a metal coating to samples, to separate the conductivity and deposition problems has yielded satisfactory deposition procedures in various laboratories, but extension of this protocol to high resolution imaging of macromolecules has yet to be demonstrated. In this paper we present a review of the main results obtained in our laboratory, which illustrate the main problems encountered by investigators attempting to image metal-coated and uncoated biological specimens.     

Scanning electron microscopy of vaginal colonization.

Changes in the appearance of the vaginal epithelium of rats during the estrous cycle were seen by scanning electron microscopy. Bacterial colonization of this tissue appeared to be influenced by these changes.     

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.     

Scanning electron microscopy of four teleostean retinas.

The morphology of the retinas of the goldeye Hiodon alosoides (fam. Hiodontidae), the brook trout Salvelinus fontinalis (fam. Salmonidae), the yellow perch Perca flavescens and the walleye Stizostedion vitreum (fam. Percidae) was studied by SEM. Semi-thin plastic sections of the same retinas were also examined for comparison. Contrary to observation of earlier authors the goldeye retina was found to possess both rods and cones; in the case of the other three fishes' retinas, present observations correspond to previous ones, adding only details. SEM gives an impressive, three dimensil view of the gross surface features of the retinas. Shrinkage during the processing of the specimens for SEM, while not altering the general topography, does induce artifacts in both plexiform layers.     

Scanning electron microscopy characterization of surgical instrument damage to breast implants.

In this article, mechanisms of breast-implant failure caused by surgical instruments commonly used to perform implantation, breast biopsies, needle localization procedures, cyst aspirations, and explantation are described. Failure was artificially induced in breast-implant shells using various types of surgical instruments, including scalpels, suture needles, hypodermic needles, hemostats, and Adson forceps. Field-emission scanning electron microscopy (SEM) was used to document the morphology of the failure sites produced by these instruments. Micrographs were used to categorize failure according to a specific type of surgical instrument. SEM micrographs were also obtained on explants that failed in situ, and the morphology of the corresponding failure sites was examined. The study was designed to document a range of failure mechanisms associated with gel-filled, saline-filled, double-lumen (saline-gel), and soybean oil-filled implants. The results of the study also demonstrate that SEM can often be used to determine the cause of breast-implant failure.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy