Life Sciences Commons^™

Correlated Confocal And Intermediate Voltage Electron Microscopy Imaging Of The Same Cells Using Sequential Fluorescence Labeling, Fixation, And Critical Point Dehydration, Lee D. Peachey, Harunori Ishikawa, Tohru Murakami

Scanning Microscopy

Confocal laser scanning microscopy (CLSM) and intermediate voltage transmission electron microscopy (IVEM) each has its own particular advantages. CLSM can examine living cells, but is particularly useful when applied to cells that have been lightly fixed, permeabilized, and stained with fluorescent-labeled antibodies for localization of specific molecular species at the resolution of the light microscope while still in the hydrated state. IVEM provides much higher resolution images, but requires more drastic preparation procedures, including dehydration. This paper presents methods for combining these complementary approaches to examine exactly the same cells sequentially by CLSM and IVEM. Cells are grown in culture …

Immunocytochemistry By Electron Spectroscopic Imaging Using Well Defined Boronated Monovalent Antibody Fragments, M. M. Kessels, B. Qualmann, W. D. Sierralta

Scanning Microscopy

Contributing to the rapidly developing field of immunoelectron microscopy a new kind of markers has been created. The element boron, incorporated as very stable carborane clusters into different kinds of peptides, served as a marker detectable by electron spectroscopic imaging (ESI) - an electron microscopic technique with high-resolution potential.

Covalently linked immunoreagents conspicuous by the small size of both antigen recognizing part and marker moiety are accessible by using peptide concepts for label construction and their conjugation with Fab' fragments. Due to a specific labeling of the free thiol groups of the Fab' fragments, the antigen binding capacity was not …

In Situ Hybridization, In Situ Transcription, And In Situ Polymerase Chain Reaction, L. E. De Bault, J. Gu

Scanning Microscopy

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR …

Preparation Of Samples For Polymerase Chain Reaction In Situ, Gerard J. Nuovo

Scanning Microscopy

The purpose of this paper is to describe the key variables in sample and reagent preparation needed for successful polymerase chain reaction (PCR) in situ. Tissue or cell preparations should be fixed in a cross linking fixative, such as 10% buffered formalin, preferably from 15 to 48 hours. Tissues should be embedded in paraffin; cell preparations can be fixed when near confluence, then physically removed and processed. When possible three samples (4 μM tissue sections or 1-5000 cells) should be placed on silane coated glass slides. Digestion in pepsin (2 mg/ml) for 30 min is adequate for DNA detection …

Microscopic Analysis Of Dna And Dna-Protein Assembly By Transmission Electron Microscopy, Scanning Tunneling Microscopy And Scanning Force Microscopy, T. Müller-Reichhert, H. Gross

Scanning Microscopy

To investigate DNA and DNA-protein assembly, nucleic acids were adsorbed to freshly cleaved mica in the presence of magnesium ions. The efficiency of DNA adhesion and the distribution of the molecules on the mica surface were checked by transmission electron microscopy. In addition, various kinds of DNA-protein interactions including DNA wrapping and DNA super-coiling were analyzed using electron microscopy. In parallel, this Mg²⁺/mica method can be applied (1) to analyze embedded DNA by scanning tunneling microscopy, (2) to visualize freeze-dried, metal coated DNA-protein complexes by tunneling microscopy, and (3) to image DNA or DNA-protein interaction in air or …

Aspects Of Cryofixation And Cryosectioning For The Observation Of Bulk Biological Samples In The Hydrated State By Cryoelectron Microscopy, K. Richter

Scanning Microscopy

Cryoelectron microscopy allows the observation of hydrated samples at high spatial resolution, and it would be of great interest in biology to apply this method to cells and tissues. However, because of technical problems, the cryo-observation of frozen hydrated ultrathin sections of bulk material has not become an established method. The major limitations are due to the difficulty of achieving the vitrification of such material, and the structural deformation caused by ultrathin sectioning: 1. The vitrification of cells in a physiological environment requires high-pressure freezing. However, new results suggest that the pressure may alter the ultrastructure of the sample. 2. …

Electro-Optical Imaging Of F-Actin And Endoplasmic Reticulum In Living And Fixed Plant Cells, Nina Stromgren Allen, Marty N. Bennett

Scanning Microscopy

Confocal and video micrographs of living and fixed alfalfa roots, onion epithelial and pear pollen cells illustrate the architecture of the cytoskeleton and endoplasmic reticulum in plant cells. Fixation of plant tissues to preserve cytoplasmic structure poses special problems. When possible, emphasis should be placed on the imaging of structures in stained living cells over time. The early events that occur when Nod factors or bacteria elicit nodule formation in alfalfa roots will illustrate several approaches to plant cell fixation, staining and imaging. The first observable events after Nod factor stimulation occur in root hairs and are changes in rates …

A Scanning Electron Microscopy Study Of Taxus Leaves As Related To Taxonomy, G.A. Strobel, W. M. Hess

Scanning Microscopy

Scanning electron microscopy, when applied to the surfaces of the needles of Taxus spp. (yew) revealed features that appear useful in the taxonomy of this tree species which yields the important anticancer drug, taxol. For instance, all of the four North American species have 3-5 rows of stomata on one-half of the abaxial leaf surface, whereas all of the others, including those from Europe and Asia, have 7-10 rows of stomata. The appearance of individual or fused papilliform epidermal cells and their arrangement on the leaf surface also is a feature that varies between species. Patterns of wax formation appeared …

The Chromatin Structure Of Well-Spread Demembranated Human Sperm Nuclei Revealed By Atomic Force Microscopy, M. J. Allen, E. M. Bradbury, R. Balhorn

Scanning Microscopy

The fundamental structure formed when genomic DNA is packaged by protamine in the human sperm nucleus still remains essentially unresolved. It is known that the binding of protamine, a small arginine-rich protein, to DNA generates a large dense, hydrophobic complex making the sperm chromatin structure difficult to study microscopically. To visualize the internal nuclear structures, isolated human sperm nuclei were swollen extensively in saline buffer using only a reducing agent. The nuclei were swollen during deposition onto coverglass and then imaged in the atomic force microscope (AFM). The two main results obtained from imaging individual well-spread nuclei indicate that native …

Atomic Force Microscopy Of Humic Acids, A. Ikai, R. Osterberg

Scanning Microscopy

Atomic force microscopic (AFM) images of humic acids show discrete, globular particles, where particles of the order of magnitude 100 to 300 nm dominate the image fields; the humic acids had been grown to a steady state at pH 5.0. The AFM data are consistent with our previously reported small-angle neutron scattering (SANS) study done under similar conditions. In further agreement, the cluster-cluster interactions shown in our previous SANS study may have their counterparts in closely interacting particles appearing as twin particles in the AFM images.

Preparation Of Cultured Smooth Muscle Cells From Human Myometrium For X-Ray Microanalysis, Jarin Hongpaisan, Godfried M. Roomans

Scanning Microscopy

Methodological aspects of the use of X-ray microanalysis in physiological and pharmacological experiments on cultured myometrial cells were investigated. Cultured human myometrial cells were grown from biopsies after detaching the fibroblasts. Of the cultured cells, 95-98% showed desmin-like immunoreactivity. Transmission electron microscopy showed that subcultured cells were different from myometrial cells in situ. The effects of washing the cells to remove external salt-rich medium were investigated. All solutions removed the external medium, resulting in lower concentrations of Na and Cl. In the cells washed with 0.3 M mannitol, most of the elemental concentrations were significantly lower than in their …

Morphological And Chemical Properties Of Plastic Residues In Composts, Charles R. Krause, Leona E. Horst, Harry A. J. Hoitink

Scanning Microscopy

The relative quantity of plastic film residues and other man-made materials in composts prepared from municipal solid wastes (MSW), biosolids, yard wastes, and pine bark was evaluated utilizing light and scanning electron microscopy equipped with energy dispersive X-ray analyzer. MSW composts screened through a 4 mm screen contained significant but highly variable quantities of plastic film residues and other man-made materials that escaped detection with the unaided eye. The other composts were free of such particles. We conclude that the database for evaluation of man-made materials in MSW composts is inadequate and must be developed further.

Relative Intranuclear Magnesium And Phosphorus Contents In Normal And Tumor Cells Of The Human Thyroid Gland As Revealed By Energy-Dispersive X-Ray Microanalysis, Geza L. Lukacs, Imre Zs.-Nagy, Janos Steiber, Ferenc Gyori, Gyorgy Balazs

Scanning Microscopy

Energy dispersive X-ray microanalysis was performed on altogether 42 surgically removed tissue specimens of 32 patients, which were taken either from intact thyroid parts or various histopathologically verified tumors of the thyroid gland. The tissue specimens were processed with the freeze-fracture-freeze-drying technique and then analyzed in the so-called bulk specimen form. The studies were carried out during the years 1980-81, when intranuclear monovalent ionic composition was studied in detail. From the retained total elemental peak list, it was possible to calculate retrospectively the relative intranuclear Mg and P contents. The data processed by nested (hierarchical) analysis of variance show that …

Imaging The Electrocyte Of Torpedo Marmorata By Scanning Force Microscopy, L. I. Pietrasanta, A. Schaper, G. Q. Fox, F. J. Barrantes, T. M. Jovin

Scanning Microscopy

Scanning force microscopy (SFM) and scanning electron microscopy (SEM) were used to examine the tissue structure of the electric organ of Torpedo marmorata in air and in liquid after applying fracturing and cryosectioning techniques and chemical fixation. The electric organ is organized in columns of stacked electrocytes, arranged in a honeycomb pattern. The columns were cut along a plane normal to the cell stack and thin sections were transferred to polylysine coated glass coverslips. The polarity of the electrocytes was made apparent by immunofluorescence microscopy directed to different domains of the acetylcholine receptor (AChR), thus revealing the innervated face of …

Hair Bundle Morphology On Surviving Hair Cells Of The Chick Basilar Papilla Exposed To Intense Sound, J. S. Erulkar, D. A. O'Brien, J. C. Saunders

Scanning Microscopy

Exposure to intense sound produces a well-defined "patch" lesion on the chick basilar papilla in which 30-35% of the short hair cells are lost. The present study compares various aspects of sensory hair bundle morphology on surviving hair cells in the patch lesion with hair bundles from matched locations on nonexposed control papilla immediately after removal from the exposure and 12-days post exposure. The height and thickness of the hairs, the total number of hairs in the bundle, the width of the bundle, and the area and perimeter of the apical surface of the hair cell were quantified from scanning …

Paleomicrobiological Study In Dental Calculus: Streptococcus Mutans, A. Linossier, M. Gajardo, J. Olavarria

Scanning Microscopy

Morphological types of bacterial remains preserved in ancient tartar of teeth from extinct human groups, which included some communities of coastal gatherers, fishermen, hunters, and farmers, and those practicing a mixed economy, were analyzed. Previous studies have shown the presence of bacteria in ancient tartar. The aim of this work was to determine whether Streptococcus mutans was present in ancient populations (500-12,000 years old). Teeth samples were from ancient skulls obtained from different anthropological collections: the north and south of Chile (before the Spanish conquest), Palencia, Spain, and an eastern Mediterranean region (Levant). Optical microscopy showed Gram positive and Gram …

Video Rate Confocal Laser Scanning Reflection Microscopy In The Investigation Of Normal And Neoplastic Living Cell Dynamics, Pavel Vesely, Alan Boyde

Scanning Microscopy

The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the live or taped video image was achieved at zoom factor 5, giving a display monitor field …

Secondary Electron Emission From Simple Metals: Comparative Studies For Al, Mg, And Be, M. Rosler

Scanning Microscopy

In the secondary electron emission (SEE) from solids, the role of different excitation processes is now as ever of special interest from both the theoretical and the experimental points of view. Depending on the primary energy, the relative importance of different excitation mechanisms related to conduction as well as core electrons will be discussed for different simple metals. So far, first principles results are available only for Al for primary energies up to 10 keV. Starting from a microscopic description of the SEE based on the transport equation formalism, calculations were performed for other nearly-free-electron metals (Mg, Be) up to …

Simultaneous Identification Of A Specific Gene Protein Product And Transcript Using Combined Immunocytochemistry And In Situ Hybridization With Non-Radioactive Probes, Gwen V. Childs

Scanning Microscopy

Simultaneous identification of messenger RNA (mRNA) and proteins in the same cells or tissues is a valuable tool to help the cell biologist evaluate the cell secretory cycle. Some cells may produce the mRNA and delay the production of the proteins. Alternatively, the proteins may be rapidly secreted. Other cells may produce both in sequence within the same time frame. Because of this difference, some cells can only be identified by their mRNA product. Others may have both products. This presentation describes a non-radioactive approach to the detection of both products with dual-peroxidase labeling protocols in use in this laboratory …

Deposition Of Supercoiled Dna On Mica For Scanning Force Microscopy Imaging, B. Samori, I. Muzzalupo, G. Zuccheri

Scanning Microscopy

The deposition of DNA molecules on mica is driven and controlled by the ionic densities around DNA and close to the surface of the substrate. Dramatic improvements in the efficiency and reproducibility of DNA depositions were due to the introduction of divalent cations in the deposition solutions. The ionic distributions on DNA and on mica determine the mobility of adsorbed DNA molecules, thus letting them assume thermodynamically equilibrated conformations, or alternatively trapping them in non-equilibrated conformations upon adsorption.

With these prerequisites, mica does not seem like an inert substrate for DNA deposition for microscopy, and its properties greatly affect the …

Fluorescence Imaging And Spectroscopy Of Biomaterials In Air And Liquid By Scanning Near-Field Optical/Atomic Force Microscopy, H. Muramatsu, N. Chiba, K. Nakajima, T. Ataka, M. Fujihira, J. Hitomi, T. Ushiki

Scanning Microscopy

We have developed scanning near-field optical/atomic force microscopy (SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fiber cantilever is 15-40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/ AFM system contains photon counting system and polychrometer/intensified coupled charge devise (ICCD) system to observe fluorescence image and spectrograph of micro areas, respectively. Cultured cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-keratin antibody or FITC-labeled phalloidin after treatment with Triton X-100. Fluorescence and topographic images were obtained in air and …

Electrical Characterization Of Submicrometer Silicon Devices By Cross-Sectional Contact Mode Atomic Force Microscopy, P. De Wolf, T. Trenkler, T. Clarysse, M. Caymax, W. Vandervorst, J. J. Snauwaert, L. Hellemans

Scanning Microscopy

Two contact mode atomic force microscopic (AFM) techniques under ambient conditions are presented for the electrical evaluation of cross sectioned silicon devices. In the first technique, a conductive AFM tip is used as a voltage probe to determine the local potential distribution on the cross section of a silicon device under operation. The electrical potential is measured simultaneously with the surface topography with nanometer resolution and mV accuracy, offering an easy way of correlating topographic and electrical features. A second method, nanometer spreading resistance profiling (nano-SRP), performs localized spreading resistance measurements to determine the spatial distribution of charge carriers in …

Nucleic Acid Detection By In Situ Molecular Immunogold Labeling Procedures, Marc Thiry

Scanning Microscopy

We have recently combined immunogold labeling procedures with molecular biology methods to pinpoint the precise locations of nucleic acids in biological material at the ultrastructural level. These new immunocytological approaches involve the incorporation of labeled nucleotides in the nucleic acids present at the surface of ultrathin sections prior to immunogold labeling. The antibodies used recognize a nucleoside analogue (bromodeoxyuridine) or a hapten (biotin) employed to label nucleotides. Examples of high-resolution detection include DNA or RNA present in different substructures of cell nuclei, and in particular, in adenovirus-induced intranuclear regions of HeLa cells. In addition to being highly sensitive and specific, …

Freeze-Dried Human Leukocytes Stabilized With Uranyl Acetate During Low Temperature Embedding Or With Oso4 Vapor After Embedding, L. Edelmann, A. Ruf

Scanning Microscopy

Two new simple stabilization procedures for freeze-dried biological material are introduced which are compatible with low temperature embedding (LTE) in Lowicryl. The first method uses a Lowicryl K11M/HM20 mixture supplemented with 0.3% uranyl acetate for LTE. For the second method polymerized Lowicryl blocks containing the freeze-dried material are exposed to OsO₄ vapor which penetrates into the Lowicryl block and stabilizes the embedded specimen. The quality of structural preservation is demonstrated with human leukocytes.

Towards In Situ Atomic Force Microscopy Imaging Of Biofilm Growth On Stainless Steel, D. T. Goddard, A. Steele, I. B. Beech

Scanning Microscopy

Atomic force microscopy (AFM) has been used to visualise the formation of bacterial biofilms on polished surfaces of 316 stainless steel. Imaging under ambient conditions revealed both the bacterial cells and the matrix of exopolymeric substances (EPS). These images exhibited good resolution with cell surface features as small as 30 nm distinguishable. In situ imaging was also carried out, and although the resolution was considerably reduced, images revealing the process of bacteria division have been obtained.

X-Irradiation-Induced Changes Of The Prelysosomal And Lysosomal Compartments And Proteolysis In Ht-29 Cells, Z. Somosy, A. Takáts, G. Bognár, A. L. Kovács, Á. Telbisz, A. Rácz, J. Kovács, G. J. Köteles

Scanning Microscopy

As a consequence of external and internal ionizing radiation, lysosome-like bodies have been observed to increase both in size and number in some cell types. We investigated this process by morphological methods (electron microscopy, cationized ferritin uptake, acid phosphatase histochemistry, morphometry) in cultured HT-29 cells. In parallel with these studies, we measured the rate of protein degradation on the basis of 14C-valine release from prelabeled cellular proteins. We found that at 2 and 4 Gy doses of X-irradiation the volume of the vacuolar (probably lysosomal) compartment increased without detectable changes of acid phosphatase activity. A 2 Gy irradiation dose did …

Ion And Electron Emission From Liquid Metal Sources, H. Niedrig

Scanning Microscopy

In liquid metal ion sources, the emission is located at the apex of a liquid cone (the often so-called Taylor cone), formed by electrostatic forces and surface tension. Reversal of the extraction voltage polarity results in electron emission from the liquid metal surface. For small apex radii, ≤ 1 μm, steady field emission of electrons has been observed, whereas for apex radii ≥ 10 μm, explosive pulsed emission occurs. Since the onset voltage for electron emission has been found to be considerably lower than the critical voltage for the formation of the Taylor cone, it has been concluded that dc …

A Comparison Of The Renal Structures Of The Anaconda And The Ball Python, H. Ditrich

Scanning Microscopy

The renal vascular system of the Ball python (Python regius) and the anaconda (Eunectes noteus; Serpentes - Squamata) has been investigated using light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) of vascular corrosion casts and critical-point dried non-corroded specimens. The average glomerular diameters of these two species differ significantly (anaconda: 59.1 μm, python: 124.3 μm). Also, the relative proportions of the renal tubules are different. These findings can be related to the different habitats of the two species (aquatic versus terrestrial environment).

Atomic Force Microscopy Investigation Of Radiation-Induced Dna Double Strand Breaks, D. Pang, G. Popescu, J. Rodgers, B. L. Berman, A. Dritschilo

Scanning Microscopy

We have used atomic force microscopy (AFM) to study radiation-induced DNA double strand breaks. Double-stranded plasmid DNA was irradiated with 18-MeV electrons in aqueous buffer, using a medical linear accelerator. Doses of 50, 100, 150, and 200 Gy were delivered to DNA samples, and atomic force microscopy was used to measure the length of each DNA fragment. From these measurements, we obtained the average length of the irradiated DNA for each sample and found a linear-quadratic relationship between the average length and radiation dose.

A Micro-Raman Spectroscopic Study Of Hydrazine-Treated Human Dental Calculus, H. Tsuda, W. L. Jongebloed, I. Stokroos, J. Arends

Scanning Microscopy

Hydrazine has been used to remove organic components and to isolate the mineral(s) from human calculus. Micro-Raman measurements were performed on the mineral phase. After the hydrazine-treatment, not only a large reduction in fluorescence but also an increase in Raman signal was observed. The treatment was essential in minimizing thermally-induced chemical changes which could otherwise occur to the original calculus mineral due to the intense laser light. The Raman spectral features of the mineral were nearly all identical among the Raman spectra obtained at many randomly-selected sites by the micro-Raman microbe with a lateral resolution of approximately 1 μm, and …