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PubMed2005

A review of scanning probe microscopy investigations of liposome-DNA complexes.

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.

DNA↗

PubMed1985

Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

Animals↗

PubMed1986

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy.

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.

Adenosine Triphosphate↗

PubMed1986

Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

Animals↗

PubMed1991

Lateral diffusion and retrograde movements of individual cell surface components on single motile cells observed with Nanovid microscopy.

A recently introduced extension of video-enhanced light microscopy, called Nanovid microscopy, documents the dynamic reorganization of individual cell surface components on living cells. 40-microns colloidal gold probes coupled to different types of poly-L-lysine label negative cell surface components of PTK2 cells. Evidence is provided that they bind to negative sialic acid residues of glycoproteins, probably through nonspecific electrostatic interactions. The gold probes, coupled to short poly-L-lysine molecules (4 kD) displayed Brownian motion, with a diffusion coefficient in the range 0.1-0.2 micron2/s. A diffusion coefficient in the 0.1 micron2/s range was also observed with 40-nm gold probes coupled to an antibody against the lipid-linked Thy-1 antigen on 3T3 fibroblasts. Diffusion of these probes is largely confined to apparent microdomains of 1-2 microns in size. On the other hand, the gold probes, coupled to long poly-L-lysine molecules (240 kD) molecules and bound to the leading lamella, were driven rearward, toward the boundary between lamelloplasm and perinuclear cytoplasm at a velocity of 0.5-1 micron/min by a directed ATP-dependent mechanism. This uniform motion was inhibited by cytochalasin, suggesting actin microfilament involvement. A similar behavior on MO cells was observed when the antibody-labeled gold served as a marker for the PGP-1 (GP-80) antigen. These results show that Nanovid microscopy, offering the possibility to observe the motion of individual specific cell surface components, provides a new and powerful tool to study the dynamic reorganization of the cell membrane during locomotion and in other biological contexts as well.

Adenosine Triphosphate↗

PubMed1975

Scanning electron microscopy of tobacco mosaic virus-labeled lymphocyte surface antigens.

The study of surface antigen by immunoelectron microscopy has been hampered by the fact that thin sections of cells provide only a view of the cell perimeter in an essentially two- dimensional fashion. Although the reconstruction of the entire cell from serial sections has been accomplished (1), it remains too exacting a technique and will find only exceptional application. Carbon-platinum replicas (2) allow the inspection of larger surface areas and therefore are better suited for studying the distribution of antigens (3). But since only relatively smooth surfaces will yield stable replicas, cells with large numbers of microvilli are not amenable to this technique. Despire its limited resolution, scanning electron microscopy (SEM) seems to be the method of choice because it can provide a view of almost half of the surface of a cell close to its natural configuration, particularly after critical point or freeze drying (4, 5). Immunological-labeling methods have not yet been routinely applied to SEM although both latex spheres (6) and hemocyanin (7) have been used with some success. The optimal visual marker should possess the following properties: be of a distinctive shape, chemically stable, and have per se a low binding affinity for cell surfaces. Tobacco mosaic virus (TMV), a marker with which we are familiar in transmission electron microscopy (8), seems to meet these demands; it has rod-like shape and defined dimensions (15 x 300 nm) and in addition it can easily be distinguished from surface microvilli. As the hybrid antibody technique (9) is also applicable to TMV, we have attempted to combine such immunological labeling with SEM. We present evidence that surface antigens can indeed be visualized by SEM, using the TMV marker in conjunction with the hybrid antibody technique.

Animals↗

PubMed2006

Scanning electron microscopy of thyroid cells under fully hydrated conditions--a novel technique for a seasoned procedure: a brief observation.

Technical information for handling fine-needle aspiration samples from thyroid lesions for WETSEM electron microscopy is presented. The use of wet SEM technology maintains cytological features of the thyroid cells, in the atmospheric electronic microscope chamber without the need for solidification. Images are presented from normal and pathological thyroid specimens showing subcellular elements unavailable to the cytopathologist by light microscopy. Of 24 samples, 18 were adequate for clinical evaluation. In 16 of these 18 specimens, we could find features compatible with the final histological or cytological diagnosis (post-hoc). In two cases, the cell features were too unique to be interpretable. Because this procedure is relatively simple, there is potential for the use of this technology as an adjunct to light microscopy in clinical and research settings.

Adult↗

PubMed1997

Dermatopathology via a still-image telemedicine system: diagnostic concordance with direct microscopy.

OBJECTIVE: To determine the concordance of dermatopathology diagnosis by still-image telemedicine technology and direct microscopy. MATERIALS AND METHODS: Skin specimens (N = 79) were examined by a dermatopathologist using a still-image phone system, and the diagnoses were compared with those made by the same dermatopathologist 1 year earlier by direct microscopy. The telemedical diagnoses were reached first without, and then with, patient histories. RESULTS: When the patient history was available, identical diagnoses were made in 66 of the 79 cases (84% concordance rate). Without patient history, the concordance rate was 80%. The diagnostic concordance rate for the diagnosis of benign nevocytic nevi, inflammatory diseases, and benign and malignant non-squamous cell carcinoma neoplasms was statistically significantly greater than the concordance rate for the diagnosis of squamous cell carcinoma and squamous cell carcinoma in situ (P = 0.005). CONCLUSIONS: The diagnostic concordance rate achieved by teledermatopathology using a still-image phone system fell short of the 99% intraobserver diagnostic concordance rate using direct microscopy.

Humans↗

PubMed1975

Periodic acid-Schiff-light green stain to detect glomerular protein deposits by routine light microscopy.

A simple, easily reproducible periodic acid-Schiff-light green stain (PAS-LG) for the detection of glomerular protein deposits by routine light microscopy is described. The deposits are selectively stained a deep blue and contrast sharply with the staining of adjacent glomerular structures. Correlation with immunofluorescent and electron microscopy has shown that it is possible with this stain to categorize accurately a large variety of glomerular lesions by light microscopy alone.

Antigen-Antibody Complex↗

PubMed1981

High resolution light microscopy in renal pathology.

Seven hundred renal specimens embedded in epoxy resins and stained with polychromatic stains were compared with paraffin sections stained with Hematoxylin and Eosin (H & E), Perodic and Schiff, (PAS), silver, and trichrome stains. High resolution of light microscopy, never in wax histopathology, may be obtained by the use of plastic embedding and polychromatic stains. Cell boundaries, intracellular organelles, basement membranes, different cellular types, apposition of different substances, and other pathologic changes were readily recognized in a single P + P (Plastic section stained with Polychromatic stains) section, whereas paraffin sections usually needed special stains. The same plastic block may be used for transmission electron microscopy. Slightly elevated cost, special training of the technician and pathologist, and some few remaining technical difficulties are the disadvantages of this method. High resolution light microscopy methods are recommended for routine renal biopsies.

Basement Membrane↗

PubMed1984

Unnecessary microscopy in routine urinalysis.

The consequence of omitting urine sediment microscopy in specimens with normal physicochemical testing was assessed in a retrospective review of laboratory and clinical data in 1,000 patients. The pathologic states of clinical significance most likely to be overlooked were Trichomonas infection and occasional cases of significant bacteriuria. However, the medical benefit of performing urine microscopy in these two groups of patients was not clear. The authors cautiously recommend reserving microscopy for urine specimens that show physicochemical abnormalities.

Bacteriuria↗

PubMed1989

Histological diagnosis of right ventricular dysplasia: a role for electron microscopy?

Right ventricular dysplasia is a rare cardiomyopathy predominantly involving the right ventricle. We report eight cases studied with light and electron microscopy. By light microscopy, we found the typical replacement of myocardium with mature adipose tissue where only scattered degenerated myocytes wrapped in thin fibrous sheets are seen. Electron microscopy shows abnormal intercalated discs in both the right and left ventricles: intercalated discs are attenuated and flattened with decreased Z band material (desmin). These changes, not previously reported, may be useful for diagnosing ARVD.

Adipose Tissue↗

PubMed1982

Detection by immune electron microscopy of the Snow Mountain agent of acute viral gastroenteritis.

An extensive outbreak of acute gastroenteritis of unknown etiology occurred at Snow Mountain, Colorado, in December 1976. Virus-like particles, 27 nm in diameter, were observed by electron microscopy in two of five stool specimens from individuals in the outbreak. Oral administration of a filtrate from one of the specimens induced disease in nine of 12 normal volunteers. Experimentally induced illness was similar to that observed during the outbreak. Stool specimens examined by immune electron microscopy revealed 27-nm virus-like particles in three of nine ill volunteers at the onset of illness. When the particles were used as a source of antigen, increases in levels of serum antibody were demonstrated in persons with either experimentally induced or naturally occurring illness. Therefore, it is likely that this virus-like particle is the etiologic agent of the Snow Mountain outbreak. The Snow Mountain agent appears to be morphologically similar to, but antigenically distinct from, the Norwalk and Hawaii agents by immune electron microscopy and may represent an additional antigenic type among the agents that resemble the Norwalk particle.

Adolescent↗

PubMed1992

Preparation and examination of flea larvae (Siphonaptera) by light and electron microscopy.

Techniques are described for making permanent slide-mounted specimens. Larvae were thoroughly macerated in KOH to remove soft internal tissue, yielding specimens with enhanced transparency and allowing examination of most of the exoskeleton in a single larva. Fatty material was removed in acetone to eliminate confusion of fat globules with sensilla in the cuticle. The more sclerotized exoskeletal elements, such as integumentary scales and the body sclerites together with their setae and sensilla, were highlighted by staining with carbol fuchsin. Resolution of numbers and arrangement of setae, especially those on the dorsum of the head capsule, in the anal comb, and on the anal mounds, was greatly facilitated by mounting larvae in dorsal orientation. Clearer views of the antennae and mouthparts were obtained, in orientations seldom achieved in whole mounts, by dissecting the head capsule. Discrimination of many taxonomically valuable features was enhanced by the use of phase-contrast microscopy. Interpretation of structures noted in light microscopy was complemented using scanning electron microscopy. The latter allowed differentiation between surface and internal cuticular structures.

Animals↗

PubMed2004

Application of real-time confocal laser scanning microscopy to observe living cells in tissue specimens.

Fluorescence microscopy imaging has developed into an important tool for the study of cell structure and function in cell biology. This non-invasive technique permits the characterization, localization and qualitative quantification of free ions, messengers, pH, voltage and other molecules in living cells. The regulation of cytosolic Ca2+ homeostasis is essential for cells. However, most investigations have used cultured or isolated cells as an experimental model and, consequently, provide only limited insight into the mechanisms that operate in tissue in situ. More useful information could be obtained by studying intact tissue specimens. The calcium dynamics of some tissue specimens, such as arteriole smooth muscle cells, supra cervical ganglia and peripheral nerve bundles, were analysed in this study. Real-time confocal microscopy revealed that individual cells exhibited different [Ca2+]i dynamics and the responses to transmitters/modulators were heterogeneous. It is important that the confocal microscopes have good detection performances, due to the reduction of stray light. We conclude that real-time confocal microscopy is a useful tool for investigating structural and functional changes of cells in living tissues, although suitable tissue-preparation is important for these measurements.

Animals↗

PubMed1992

Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements.

Atomic force microscopy (AFM) was used to image reovirus double stranded RNA (dsRNA) deposited from diluted buffer solution onto a chemically treated mica surface. This procedure allows AFM images of dsRNA molecules to be obtained with a quality close to that obtained with conventional electron microscopy. The length of the molecules were measured directly on a computer display using the digitally acquired images. The lengths of the molecules varied between 0.2 and 1.8 microns. Statistical analysis showed a multimodal distribution with clear maxima at 0.4, 0.65 and 1.05 microns. These data are in a good agreement with those obtained by electron microscopy and gel electrophoresis.

Microscopy, Scanning Tunneling↗

PubMed2001

Assessment of haematuria: automated urine flowmetry vs microscopy.

BACKGROUND: Microscopy of the urine sediment may be a useful method in the distinction between a glomerular and a non-glomerular source of urinary bleeding. However, microscopic techniques are time consuming and hampered by inter-observer variations. In the present study we have therefore compared bright-field microscopy with automated urine flowmetry (Sysmex UF-100), examining their ability to differentiate between glomerular and non-glomerular haematuria. METHODS: Fresh urine samples were obtained from 112 patients with a well-defined, single cause of a positive dipstick test. Their urine specimens were examined within 4 h in a blinded manner. Of them, 79 specimens had a positive dipstick for blood and thus could be evaluated for haematuria. RESULTS: The Sysmex UF-100 had a sensitivity and specificity of 0.83 and 0.94 respectively in detecting non-glomerular bleeding. The positive and negative predictive values were 0.95 and 0.78 respectively. The corresponding values of microscopy were 0.79 and 0.90 respectively, and 0.93 and 0.74 respectively. CONCLUSIONS: Automated flowmetry can be used in the distinction between glomerular and non-glomerular haematuria.

Adult↗

PubMed1986

Meiotic chromosome studies and synaptonemal complex analyses by light and electron microscopy in 47 infertile or sterile males.

Mitotic and meiotic chromosome studies and synaptonemal complex analyses by light and electron microscopy have been carried out in a selected series of 47 infertile or sterile males with highly abnormal seminograms, affecting the number of spermatozoa, their morphology and/or motility. In 46 cases, the karyotype was 46,XY. One patient had a 13/14 translocation. With the exception of the patient with a 13/14 translocation, and three patients with desynapsis (8.5%), all other cases showed either normal or absent metaphase I figures. However, synaptonemal complex analysis by light and electron microscopy demonstrated the presence of pairing anomalies (desynapsis, fragmented or irregular synaptonemal complexes) in 31.9% of the patients studied. The total number of synaptic anomalies observed (40.4%) is higher than in a former light microscopy study of 111 infertile or sterile patients (28.8%) probably because the higher resolution of the electron microscope permits the characterization of some anomalies that cannot be detected with the light microscope. The electron microscope should therefore be used in all cases in which the light microscope provides doubtful results.

Chromosomes, Human↗

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