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At least 955 records · Page 53Linked to original sources
PubMed2006

High-resolution intravital NADH fluorescence microscopy allows measurements of tissue bioenergetics in rat ileal mucosa.

OBJECTIVES: NADH fluorescence microscopy has been used as an index of the metabolic state of tissue but is associated with various obstacles such as low spatial resolution and quenching effects of blood pigments that prevent reliable monitoring of tissue bioenergetics. The objective of this study was to develop a system to monitor tissue bioenergetics in vivo using NADH fluorescence microscopy in the rat ileal mucosa. MATERIALS AND METHODS: Using an inverted microscope with an epifluorescence unit and an intensified charge-coupled device camera, NADH fluorescence images were visualized. Fluorescence intensity was measured of beta-NADH solutions at varying concentration (n = 6) and pH (n = 3) and in ex vivo (n = 6) and in vivo (n = 6) preparations of ileal mucosa of Sprague-Dawley rats anesthetized with isoflurane. RESULTS: Intravital fluorescence microscopy reveals a map of the microcirculation that permits visualization of NADH fluorescence and intercapillary areas. The system was adjusted so a linear relationship between physiological concentrations of beta-NADH and fluorescence was achieved (r(2) = 0.98, p < .0001). Decreasing the pH of the solution had no effect on fluorescence intensity and fluorescence intensity in an anoxic ex vivo ileal segment was similar to that of the in vivo ileum after ischemia. Ischemia also resulted in spatial heterogeneity that was abolished by the addition of a 550-nm LP filter. CONCLUSIONS: With this system, intravital NADH fluorescence microscopy provides the high resolution necessary to reliably monitor tissue bioenergetics in the rat ileal mucosa.

Animals↗
PubMed2005

Localization and movement of mineral oil in plants by fluorescence and confocal microscopy.

Fluorescence and confocal laser scanning microscopy were explored to investigate the movement and localization of mineral oils in citrus. In a laboratory experiment, fluorescence microscopy observation indicated that when a 'narrow' distillation fraction of an nC23 horticultural mineral oil was applied to adaxial and opposing abaxial leaf surfaces of potted orange [Citrus x aurantium L. (Sapindales: Rutaceae)] trees, oil penetrated steadily into treated leaves and, subsequently, moved to untreated petioles of the leaves and adjacent untreated stems. In another experiment, confocal laser scanning microscopy was used to visualize the penetration into, and the subsequent cellular distribution of, an nC24 agricultural mineral oil in C. trifoliata L. seedlings. Oil droplets penetrated or diffused into plants via both stomata and the cuticle of leaves and stems, and then moved within intercellular spaces and into various cells including phloem and xylem. Oil accumulated in droplets in intercellular spaces and within cells near the cell membrane. Oil entered cells without visibly damaging membranes or causing cell death. In a field experiment with mature orange trees, droplets of an nC23 horticultural mineral oil were observed, by fluorescence microscopy, in phloem sieve elements in spring flush growth produced 4-5 months and 16-17 months after the trees were sprayed with oil. These results suggest that movement of mineral oil in plants is both apoplastic via intercellular spaces and symplastic via plasmodesmata. The putative pattern of the translocation of mineral oil in plants and its relevance to oil-induced chronic phytotoxicity are discussed.

Biological Transport↗
PubMed1995

Optical microscopy. 3. Tracking fluorescently labeled neurons in developing brain.

For decades, time-lapse microscopy has been used to track dynamic events associated with biological phenomena. Time-lapse studies of the developing nervous system have been restricted to analysis of dissociated cell cultures or of a series of static images from living organisms. The advent of new fluorescent dyes and video imaging technology has produced novel views of the behavior of neurons in the context of the developing nervous tissue, such as migrations within and away from proliferative zones and navigation of axonal processes to synaptic targets. After fixation of the tissue preparation, time-lapse monitoring can be followed by other analytical techniques and forms of microscopy, e.g., immunocytochemistry or electron microscopy, producing information on the interactions of individual cells whose behavioral histories are known. The power of video time-lapse microscopy of living brain tissue lies in the firsthand documentation of developmental patterning, which in turn can serve as an experimental assay.

Animals↗
PubMed2003

Comparison of confocal biomicroscopy and noncontact specular microscopy for evaluation of the corneal endothelium.

PURPOSE: To compare the clinical efficacy of confocal biomicroscopy with that of noncontact specular microscopy for the evaluation of the corneal endothelium. METHODS: The corneal endothelium was examined in 14 normal subjects (28 eyes) and in 6 patients (11 eyes) with Fuchs corneal endothelial dystrophy using a noncontact specular microscope (SP-2000P, Topcon, Japan) and a confocal biomicroscope (ConfoScan, Tomey, Japan). The images and the calculated densities of corneal endothelial cells obtained by the 2 techniques were compared. RESULTS: For normal subjects, the images of corneal endothelial cells obtained by the 2 techniques were almost identical, although the density of these cells determined by confocal biomicroscopy (2916 +/- 334 cells/mm2) was slightly higher than that determined by noncontact specular microscopy (2765 +/- 323 cells/mm2). In contrast, whereas clear images of corneal endothelial cells, allowing the determination of cell density, were obtained for all 11 eyes of the patient group by confocal biomicroscopy, clear images were obtained for only 4 of these 11 eyes (36.4%) by noncontact specular microscopy. CONCLUSION: Both noncontact specular microscopy and confocal biomicroscopy revealed the shapes and number of endothelial cells in the normal cornea. However, for corneas with Fuchs dystrophy, clear images were obtained only by confocal biomicroscopy. Confocal biomicroscopy is thus an effective tool for evaluation of the diseased corneal endothelium.

Adult↗
PubMed2002

A new abdominal cavity chamber to study the impact of increased intra-abdominal pressure on microcirculation of gut mucosa by using video microscopy in rats.

OBJECTIVE: In experimental studies of capillary blood flow that use intravital video microscopy, organs are exposed in observation chambers implanted into the animal. In this article we describe an abdominal cavity chamber for intravital video microscopy of gut mucosa microcirculation during increased intra-abdominal pressure. DESIGN: Prospective, experimental animal study. SETTING: Research laboratory at a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: The abdominal cavity chamber was designed for implantation into the abdominal wall of rats after laparotomy, thus creating an expanded hermetic, abdominal cavity volume. Animals were assigned to three levels of intra-abdominal pressure: controls (group 1), 10 mm Hg (group 2), and 15 mm Hg (group 3). Intra-abdominal pressure was increased by intra-abdominal insufflation of gas. By using a fluorescent marker, we quantitatively assessed mucosa perfusion index, functional capillary density, red blood cell velocity, capillary diameters, and flow motion during increased intra-abdominal pressure by intravital video microscopy. Results were expressed as mean +/- SEM. Significance of differences was determined by analysis of variance and multiple comparison of means with post hoc test (*p <.05 groups vs. control;p <.05 group 3 vs. group 2). MEASUREMENTS AND MAIN RESULTS: When compared with controls, animals subjected to an intra-abdominal pressure of 10 and 15 mm Hg showed a significant stepwise decrease in mucosa perfusion index (88%, 71%*, 22%*), functional capillary density (665.4 +/- 71.7, 461.6 +/- 71.9*, 375.1 +/- 2.0*cm(-1)), and red blood cell velocity (0.50 +/- 0.04, 0.33 +/- 0.03*, 0.04 +/- 0.06*mm/sec), indicating a stepwise impairment of mucosal microcirculation. Capillary diameters and flow motion did not change with respect to intra-abdominal pressure. CONCLUSIONS: This novel animal model of intravital intestinal video microscopy that uses an abdominal cavity chamber is a feasible and sensitive experimental tool to study intestinal microcirculation during increased intra-abdominal pressure. Intra-abdominal pressure likely results in a severe impairment of mucosal microcirculation.

Abdomen↗
PubMed1977

Detection of neoplastic and preneoplastic urothelia by combined scanning and transmission electron microscopy of urinary surface of human and rat bladders.

Scanning and transmission electron microscopy reveal distinct differences between the appearance of the luminal surface membrane of normal urothelium and those of transitional cell tumours of both human and rat bladder. The luminal surface membrane of the preneoplastic rat urothelium shows features similar to those seen in the fully developed tumour. In human tumour-bearing bladders the urothelium away from the transitional cell tumour is not recognizable as being preneoplastic by light microscopy. However, by scanning or transmission electron microscopy, the luminal membrane in some areas shows changes identical to those in the preneoplastic rat bladder. These observations are in accordance with the clinical behaviour of the urothelium in patients with vesical transitional cell tumours which tend to recur in disparate site. We suggest that scanning electron microscopy of the bladder urothelium away from the tumour may be of prognostic value for indicating the subsequent biological behaviour of the urothelium.

Animals↗
PubMed1983

Video-enhanced microscopy with a computer frame memory.

Video-enhanced microscopy combined with the use of a computer frame memory extends considerably the useful range of our video enhanced contrast (AVEC) methods for polarizing, double-beam interference and differential interference contrast microscopy. Increased visual contrast is achieved by two stages of amplifications: the first optical, by using high bias retardation settings, and the second electronic. These steps are followed by a reduction of background brightness by means of a clamp voltage applied to a DC restoration circuit of the video camera. One of the limitations of the AVEC method alone is the inevitable appearance under high gain conditions of a pattern of mottle due to inaccessible dirt and defects in the lenses even of high quality. This limitation has been circumvented by storing the mottle pattern in the frame memory (frame store) and continuously subtracting it from each succeeding frame to clear the image. A major gain in image quality has resulted. In polarizing microscopy, the frame memory can be used also to subtract the image at one compensator setting from that at the equivalent setting of opposite sign, thus removing from the final image not only most of the mottle pattern but also the contrast due to the bright-field contrast. In the polarizing microscope, these manipulations of the raw video image make it possible to observe and measure the birefringence of various organelles and elements such as microtubules, intermediate filaments and bundles of as few as a half dozen actin filaments. Since scattered light is also removed from the image, features hidden from view in the unprocessed image become visible. In differential interference microscopy, the AVEC method makes visible (i.e. detectable) many linear elements and particles that are an order of magnitude smaller than the resolution limit and not visible in the optical image. Such features are inflated by diffraction, however, to Airy disk size.

Chloroplasts↗
PubMed1985

Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes.

Critical-point drying and freeze drying were compared both quantitatively and qualitatively as preparative procedures for scanning electron microscopy. Isolated hepatocytes were used as model cells. Nomarski differential interference contrast microscopy was used for light microscopic measurements of the hepatocytes in the unfixed, the glutaraldehyde fixed, the glutaraldehyde + OsO4 fixed, the critical-point dried and the freeze dried states. Critical-point dried hepatocytes were found to shrink to 38% of glutaraldehyde + OsO4 fixed volume, whereas optimal freeze dried hepatocytes (frozen in water saturated with chloroform and freeze dried at 183 K for 84 h) were found to shrink to 51% of glutaraldehyde + OsO4 fixed volume. Transmission and scanning electron micrographs of the critical-point dried cells showed well-preserved ultrastructure and surface structure. Micrographs of the freeze dried cells showed ultrastructure destroyed by internal ice crystals and surface structure destroyed by external ice crystals. Double-fixed isolated hepatocytes were shown to swell during storage in buffer and to shrink during storage after critical-point drying. For low magnification scanning electron microscopy (up to about 3000 times) both critical-point drying and freeze drying can be used. However, for high magnification scanning electron microscopy, critical-point drying is superior to freeze drying.

Animals↗
PubMed2001

Fluorescence resonance energy transfer microscopy: a mini review.

Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBPalpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides two-dimensional spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques.

CCAAT-Enhancer-Binding Proteins↗
PubMed2002

Contrast agents for confocal microscopy: how simple chemicals affect confocal images of normal and cancer cells in suspension.

Normal and malignant human cervical cancer cells were imaged in vivo with confocal, phase contrast, and brightfield microscopies. Results were compared between cells in growth medium before and after addition of acetic acid, hypertonic saline solution, toluidine blue, and Lugol's iodine. The exogenous agents changed the backscattering characteristics of the cells when measured with confocal microscopy at 808 nm. A tendency toward higher scattering was observed in treated cells. Acetic acid and toluidine blue increased the brightness of the nucleus with respect to the cytoplasm in normal and cancer cells. Hypertonic saline solution made the cytoplasm brighter than the nucleus in both types of cells. The results indicate that simple chemicals can be used to enhance confocal microscopy's ability to differentiate intracellular components, such as nuclear size and shape. This can further confocal microscopy's ability to assess disease in cells and tissues.

Acetic Acid↗
PubMedDate not supplied

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution.

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD.

Animals↗
PubMed2002

Optical projection tomography as a tool for 3D microscopy and gene expression studies.

Current techniques for three-dimensional (3D) optical microscopy (deconvolution, confocal microscopy, and optical coherence tomography) generate 3D data by "optically sectioning" the specimen. This places severe constraints on the maximum thickness of a specimen that can be imaged. We have developed a microscopy technique that uses optical projection tomography (OPT) to produce high-resolution 3D images of both fluorescent and nonfluorescent biological specimens with a thickness of up to 15 millimeters. OPT microscopy allows the rapid mapping of the tissue distribution of RNA and protein expression in intact embryos or organ systems and can therefore be instrumental in studies of developmental biology or gene function.

Anatomy, Cross-Sectional↗
PubMed1981

Improved detail in biological soft X-ray microscopy: study of blood platelets.

Improved image quality in soft x-ray contact microscopy can be obtained by examining the resist with transmission rather than scanning electron microscopy. Application of the new technique to air-dried preparations of human blood platelets reveals structures not visible in the same cells with transmission electron microscopy or when the resist is examined by scanning electron microscopy. As seen by the new technique, platelet pseudopods contain a central structure connected to a network in the platelet and dense bodies exhibit a lamellar structure.

Blood Platelets↗
PubMed1971

Microorganisms in unamended soil as observed by various forms of microscopy and staining.

A light-diffraction microscope was modified to allow sequential viewing of the microorganisms in a soil smear by transmitted, reflected, and reflected-polarized incandescent light and by reflected ultraviolet light. Observations were also made by conventional incandescent and ultraviolet transmitted-light microscopy. All results for the various forms of bright-field microscopy with stained and unstained soils were in agreement, but they differed from the results obtained for two types of ultraviolet-fluorescence microscopy. The latter proved to be nonspecific for in situ soil microorganisms. Capsule-like areas were noted surrounding many of the resident microbial cells of soil when viewed by the various forms of bright-field microscopy. These areas could not be stained or removed by a variety of treatments, but they apparently often did take up stain after in situ soil growth had been initiated. It was concluded that these areas are not capsules but may represent a structural component of nonmultiplying microbial cells in soil.

Acridines↗
PubMed2000

Partial liquid ventilation with perfluorocarbon in acute lung injury: light and transmission electron microscopy studies.

Liquid ventilation using perfluorocarbon has been shown to improve gas exchange in animal models of acute lung injury as well as in children with acute respiratory distress syndrome. This study was designed to define structural features of lung injury following partial liquid ventilation (PLV) using light and transmission electron microscopy in a rabbit model of acute respiratory distress. Animals were treated with either conventional mechanical ventilation (CMV-gas) (n = 6) or PLV (n = 5) for 4 h after the induction of acute lung injury with saline lavage. Control animals were killed after the lung injury. PLV significantly improved alveolar-arterial oxygen tension and the oxygen index compared with CMV (P < 0.05). Morphometric studies using light microscopy show less alveolar hemorrhage, less edema, and fewer hyaline membranes in the PLV group (P < 0.05). Polymorphonuclear leukocyte sequestration in lung capillaries (11.4 +/- 1.5 versus 19.2 +/- 3 x 10(8)/ml, P < 0.05, PLV versus CMV) and migration into airspaces (3.1 +/- 1.2 versus 4.5 +/- 1.1 x 10(8)/ml, P < 0.05, PLV versus CMV) were lower in the gravity-dependent lung regions. There were fewer alveolar macrophages in the PLV group compared with other groups (P < 0.05). Fluorescence microscopy analysis shows fewer type II alveolar epithelial cells in the CMV group and brighter type II cells in the PLV group. Transmission electron microscopy studies show more alveolar wall damage in the CMV group, with type II cells detached from their basement membrane with fewer surfactant-containing lamellar bodies. We conclude that quantitative histologic analysis shows less lung damage and inflammation when perfluorocarbon is combined with CMV in the management of acute respiratory distress syndrome.

Acute Disease↗
PubMed1999

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)

Animals↗
PubMed1977

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.

Candida↗
PubMed1979

A study of acid phosphatase and dipeptidyl aminopeptidase II in monodispersed anterior pituitary cells using flow cytometry and electron microscopy.

A brief historical review of cytoenzymology is presented from the time of introduction into electron microscopy to the present, where the direction for quantification of an enzyme in single cells appears most promising by fluorescent staining. First attempts are reported to quantitate acid phosphatase (AcPase) and dipeptidyl aminopeptidase II (DAP-II) in monodispersed anterior pituitary cells from lactating and postlactating rats by flow cytometry, fluorescent, and electron microscopy. 3-Hydroxy-flavone is introduced as a new fluorescent cytochemical stain for AcPase, useful in flow cytometry but of only limited use in fluorescent microscopy. Histograms for AcPase indicate a single peak of cells staining more intensely in cell preparations from postlactating over lactating animals. Histograms for DAP-II staining indicate two distinct populations of cells present in the lactating and only one in the postlactating rat anterior pituitary gland. The application of dual laser staining indicates that not all cells stain for both enzymes. Electron microscopy shows the subcellular localization of DAP-II to be limited to lytic bodies and in mammotrophic cells to some secretion granules.

Acid Phosphatase↗