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At least 1,657 records · Page 92Linked to original sources
PubMed1996

Diagnosis of microsporidial keratitis by confocal microscopy and the chromatrope stain.

PURPOSE: To illustrate the value of confocal microscopy and chromatrope stain in the diagnosis of microsporidial keratitis. METHODS: In vivo confocal microscopy was performed on a man with the human immunodeficiency virus who had severe bilateral epithelial keratitis refractory to topical antibacterial medications. The results were compared to conjunctival scrapings stained with the chromatrope-based Weber stain. RESULTS: Confocal microscopy demonstrated many small, intraepithelial opacities of the corneal epithelium, which were suggestive of Microsporidia. Results of the chromatrope stain of conjunctival scrapings confirmed the diagnosis of microsporidial keratitis. CONCLUSIONS: Rapid diagnosis allowed prompt initiation of topical fumagillin, which permitted rapid, long-term control of the symptoms of microsporidial keratitis.

AIDS-Related Opportunistic Infections↗
PubMed1998

A comparison of historic asbestos measurements using a thermal precipitator with the membrane filter-phase contrast microscopy method.

The published results and analytical methods used in a 1938 survey of the spinning area in a UK crocidolite asbestos factory have been described and re-interpreted, by comparing the method used with the current membrane filter-phase contrast microscopy (MF-PCM) method for asbestos. By good fortune, most of the original microscope, and thermal precipitator sampling heads similar to those used for sampling were available, as well as guidance from the factory inspector who collected and analysed the original samples. A textile grade crocidolite asbestos was used to generate a fibrous dust cloud whose size distribution was characterised by scanning and transmission electron microscopy and found to give a close approximation to the fibre size distributions monitored in 1938. Samples taken over the same sampling time, but at lower concentrations than originally sampled, showed that the thermal precipitator-oil immersion microscopy (TP-OI) method used at x 2000 magnification, gave higher > 5 microns long fibre counts by a factor of between 3 and 4, than the current MF-PCM method. The differences in performance could be explained by the superior resolving power of the TP-OI method for fine crocidolite fibres. Exposures of airborne asbestos fibres in the spinning area were found to be equivalent to about 20 f ml-1 for personal samples and 10 f ml-1 for area samples, but due to the high levels of ventilation on the day the samples were collected, the average levels throughout the year may have been somewhat higher.

Air Pollutants, Occupational↗
PubMed1977

Morphology of mouse epidermal cells in vitro: a scanning electron microscopy study.

Scanning electron microscopy of isolated epidermal cells from newborn mice grown in vitro showed that the cultures consisted of several morphologically different types of cells. Young cultures had many smooth, round cells while older cultures contained more cells with rough or ruffled surface and a varying number of flat, irregular cells. Probably, the various cell types corresponded to different stages of differentation (keratinization). Cultures that were grown in the presence of retinyl acetate (12.5 microgram/ml) had more round and smooth cells after several days in vitro than the controls. This could indicate that retinyl acetate delayed or altered cell differentiation. Scanning electron microscopy of the cultures consistently showed that the cells were situated at different layers. The apparent monolayer seen by phase contrast microscopy therefore seems to be an optical phenomenon due to projection of the cells onto the same plane.

Animals↗
PubMed2001

Ultrastructural morphology and localisation of cisplatin-induced platinum-DNA adducts in a cisplatin-sensitive and -resistant human small cell lung cancer cell line using electron microscopy.

Ultrastructural morphology (transmission electron microscopy) and localisation of cisplatin-induced platinum (Pt)-DNA adducts (immunoelectron microscopy) were analysed in the human small cell lung cancer cell line GLC(4) and its 40-fold in vitro acquired cisplatin-resistant subline GLC(4)-CDDP, which is characterised by, among other things, a decreased DNA platination. Immunolabelling of Pt-DNA adducts was performed with the polyclonal antibody GPt, known to detect the main Pt-containing intrastrand and interstrand DNA adducts. Morphological analysis of GLC(4) and GLC(4)-CDDP at the ultrastructural level showed cells with a high nucleus/cytoplasm ratio with the majority of nuclei containing one or more nucleoli. GLC(4)-CDDP showed, in contrast to GLC(4), an extensive Golgi apparatus and an increased number of mitochondria. DNA platination was detectable in both GLC(4) and GLC(4)-CDDP. Immunoelectron microscopy showed Pt-DNA adducts primarily in the nucleus, preferentially at loci with high-density chromatin (e.g. heterochromatin, pars granulosa around nucleoli, condensed DNA in proliferating and apoptotic cells), and in mitochondria. The level of detectable Pt-DNA adducts was cell cycle status-dependent. In both cell lines, Pt-DNA adduct levels increased from non-dividing interphase cells to dividing cells and were highest in cells undergoing apoptosis. Overall localisation of Pt-DNA adducts was comparable in GLC(4) and GLC(4)-CDDP cells.

Antibodies↗
PubMed2001

Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.

Fluorescence↗
PubMed2001

Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells.

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.

Animals↗
PubMed2002

Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy.

Sidedness and accessibility of protein epitopes in intact brush border membrane vesicles were analyzed by detecting single molecule interaction forces using molecular recognition force microscopy in aqueous physiological solutions. Frequent antibody-antigen recognition events were observed with a force microscopy tip carrying an antibody directed against the periplasmically located gamma-glutamyltrans- peptidase, suggesting a right side out orientation of the vesicles. Phlorizin attached to the tips bound to NA+/D-glucose cotransporter molecules present in the vesicles. The recognition was sodium dependent and inhibited by free phlorizin and D-glucose, and revealed an apparent K(D) of 0.2 microM. Binding events were also observed with an antibody directed against the epitope aa603-aa630 close to the C terminus of the transporter. In the presence of phlorizin the probability of antibody binding was reduced but the most probable unbinding force f(u) = 100 pN remained unchanged. In the presence of D-glucose and sodium, however, both the binding probability and the most probable binding force (f(u) = 50 pN) were lower than in its absence. These studies demonstrate that molecular recognition force microscopy is a versatile tool to probe orientation and conformational changes of epitopes of membrane components during binding and trans-membrane transport.

Animals↗
PubMed2003

Atomic force microscopy and light scattering of small unilamellar actin-containing liposomes.

Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K(+) ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures.

Actins↗
PubMed2003

Chemically resolved imaging of biological cells and thin films by infrared scanning near-field optical microscopy.

The infrared (IR) absorption of a biological system can potentially report on fundamentally important microchemical properties. For example, molecular IR profiles are known to change during increases in metabolic flux, protein phosphorylation, or proteolytic cleavage. However, practical implementation of intracellular IR imaging has been problematic because the diffraction limit of conventional infrared microscopy results in low spatial resolution. We have overcome this limitation by using an IR spectroscopic version of scanning near-field optical microscopy (SNOM), in conjunction with a tunable free-electron laser source. The results presented here clearly reveal different chemical constituents in thin films and biological cells. The space distribution of specific chemical species was obtained by taking SNOM images at IR wavelengths (lambda) corresponding to stretch absorption bands of common biochemical bonds, such as the amide bond. In our SNOM implementation, this chemical sensitivity is combined with a lateral resolution of 0.1 micro m ( approximately lambda/70), well below the diffraction limit of standard infrared microscopy. The potential applications of this approach touch virtually every aspect of the life sciences and medical research, as well as problems in materials science, chemistry, physics, and environmental research.

Animals↗
PubMed1973

Contrast in the photoelectric effect of organic and biochemical surfaces. A first step towards selective labeling in photoelectron microscopy.

The photoelectric effect can provide the physical basis for a new method of mapping organic and biological surfaces. The technique, photoelectron microscopy, is similar to fluorescence microscopy using incident ultraviolet light except that photoejected electrons form the image of the specimen surface. In this work the minimum wavelengths of incident light required to produce an image were determined for the molecules 3,6-bis(dimethylamino)acridine (acridine orange) (I), benzo[a]pyrene (II), N,N,N',N'-tetraphenylbenzidine (III), and copper phthalocyanine (IV). The photoelectron image thresholds for these compounds are 220 (I), 215 (II), 220 (III), and 240 nm (IV), all +/-5 nm. Contrast of I-IV with respect to typical protein, lipid, nucleic acid, and polysaccharide surfaces was examined over the wavelength range 240-180 nm. The low magnification micrographs exhibited bright areas corresponding to I-IV but dark regions for the biochemical surfaces. The high contrast suggests the feasibility of performing extrinsic photoelectron microscopy experiments through selective labeling of sites on biological surfaces.

Acridines↗
PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗
PubMed1992

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.

Base Sequence↗
PubMed1993

Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells.

A new method of fluorescence microscopy for cell imaging has been developed that takes advantage of the spatial variations of fluorescence lifetimes in single cells as a source of image contrast, and thus it is named "fluorescence lifetime imaging microscopy (flimscopy)". Since time-resolved fluorescence measurements are sensitive to molecular dynamics and interactions, flimscopy allows the molecular information to be visualized in single cells. In flimscopy measurements, several (nanosecond) time-resolved fluorescence images of a sample are obtained at various delay times after pulsed laser excitation of the microscope's entire field of view. Lifetimes are calculated pixel-by-pixel from these time-resolved images, and the spatial variations of the lifetimes are then displayed in a pseudocolor format (flimscopy image). The total data acquisition time needed to obtain a flimscopy image with the diffraction-limited spatial resolution (approximately 250 nm) is decreased to just approximately 30 s for approximately 300 fluorescent molecules/micron2. This was achieved by developing a high-frequency (400 kHz) nanosecond-gating (9 ns full width at half height)-signal accumulation system. This technique allows the extent of resonance energy transfer to be visualized in single living cells, and is free from the errors due to variations in path length, light scattering, and the number of fluorophores that necessitate complex corrections in steady-state microfluorometry and fluorescence ratio imaging microscopy. Flimscopy was applied here to observe the extent of fusion of individual endosomes in single cells. Results revealed the occurrence of extensive fusion between primary endocytic vesicles and/or sorting endosomes, thereby raising the possibility that the biogenesis of sorting endosomes involves multiple fusions of primary endocytic vesicles.

Animals↗
PubMed1995

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.

Aluminum Silicates↗
PubMed1996

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.

Biophysical Phenomena↗
PubMed1998

Direct detection of domains in phospholipid bilayers by grazing incidence diffraction of neutrons and atomic force microscopy.

The geometry of domains in phospholipid bilayers of binary (1:1) mixtures of synthetic lecithins with a difference in chain length of four methylene groups has been studied by two independent, direct and complementary methods. Grazing incidence diffraction of neutrons provided gel domain sizes of less than 10 nm in both the gel and the coexistence phase of the mixture, while no domains were detected for the fluid phase. For the coexistence region, the neutron data suggest that domains grow in number rather than in size with decreasing temperature. Atomic force microscopy was used to study gel phase size and shape of the domains. The domains imaged by atomic force microscopy exhibit a rather irregular shape with an average size of 10 nm, thus confirming the neutron results for this phase. The good agreement between atomic force microscopy and neutron results, despite the completely different nature of their observables, has potential for the future development of refined models for the interpretation of neutron data from heterogeneous membranes in terms of regularly spaced and spatially extended scatterers.

Calorimetry, Differential Scanning↗
PubMed1998

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.

Animals↗
PubMed2003

Scanning Near-field Optical/Atomic Force Microscopy detection of fluorescence in situ hybridization signals beyond the optical limit.

Fluorescence in situ hybridization (FISH) is widely used in molecular biological study. However, high-resolution analysis of fluorescent signals is theoretically limited by the 300-nm resolution optical limit of light microscopy. As an alternative to detection by light microscopy, we used Scanning Near-field Optical/Atomic Force Microscopy (SNOM/AFM), which can simultaneously obtain topographic and fluorescent images with nanometer-scale resolution. In this study, we demonstrated high-resolution SNOM/AFM imaging of barley chromosome (Hordeum vulgare, cv. Minorimugi) FISH signals using telomeric DNA probes. Besides detecting the granular structures on chromosomes in a topographic image, we clearly detected fluorescent signals in telomeric regions with low-magnification imaging. The high-resolution analysis suggested that one of the telomeric signals could be observed by expanded imaging as two fluorescent regions separated by approximately 250 nm. This result indicated that the fluorescent signals beyond the optical limit were detected with higher resolution scanning by SNOM/AFM.

Chromosomes↗