Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorescence Microscopy”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources
PubMedDate not supplied

Immediate placement of implants into periodontally infected sites in dogs. Part 2: A fluorescence microscopy study.

PURPOSE: Polychromatic sequence labeling of bone was used to study the effect of periodontal infection on the immediate placement of Frialit-2 implants. MATERIALS AND METHODS: In the surgical first phase, periodontitis was induced with ligatures involving the mandibular premolars of 5 mongrel dogs, and the contralateral teeth were used as controls (received only prophylaxis). After 3 months, the second phase was initiated and 40 implants were placed in the alveoli of both experimental and control teeth. During the healing period, fluorescent bone markers were injected to study bone formation around the implants. The dyes were injected in the following sequence: oxytetracycline hydrochloride at 3 days after implant placement, calcein green 4 weeks after implant placement, oxytetracycline 8 weeks after implant placement, and alizarin red S 3 days before sacrifice. Following a healing period of 12 weeks, the animals were euthanized and the hemimandibles were removed, dissected, fixed, and prepared for histomorphometic analysis of the percentage of each bone marker present. RESULTS: Fluorescence microscopy showed a similar sequence of bone remodeling (Mann-Whitney test) for both groups: experimental group, 9% bone formation at 3 days, 29% at 4 weeks, 21.6% at 8 weeks, and 52% at 12 weeks; control group, 14% at 3 days, 35.2% at 4 weeks, 32.3% at 8 weeks, and 45.8% at 12 weeks. DISCUSSION: Remodeling in both groups had similar characteristics in the degree of bone formation. CONCLUSIONS: It was concluded that periodontal disease does not affect bone remodeling around immediate implants. Although the healing in periodontally infected sites was slower initially, it reached the levels of the non-diseased sites after 12 weeks.

Alveolar Process↗
PubMed1994

Molecular organization in phospholipid monolayer domains by correlative fluorescence microscopy and electron diffraction.

Lipid monolayer is a half leaflet model for lipid bilayer, which forms the basis of biological membranes. Within a certain range of surface area per molecular of phospholipid monolayers at the air-water interface, where the compressibility was nearly infinite, two phases with different molecular packings were observable by fluorescence microscopy. Mixed-phase monolayers of L-1,2-dipalmitoyl-N-monomethyl-3-phosphatidylethanolamine [DP(Me)PE] or L-1,2-dipalmitoyl-N-dimethyl-3-phosphatidyl-ethanolamine [DP(Me)2PE] were deposited on marker grids coated with Formvar films. The molecular organization in the dark and bright fluorescent areas on the grids was investigated by low dose, selected area electron diffraction. Sharp reflection arcs, at a spacing of 4.2A and arranged in a hexagon pattern, were detected from dark domains of both lipids. A diffuse reflection ring at a spacing of 4.6A was derived from the bright background areas. Diffraction patterns were obtained from neighboring areas along selected dark domains of both lipids. The orientations of diffraction patterns from areas along smooth and curving boundaries of DP(Me)2PE domains were found to turn with the boundaries. In the branching domains of DP(Me)PE, the orientations of diffraction patterns indicated that the branches were formed by twinning. Electron diffraction thus provides an unique way to sample the local molecular packing order and orientation within individual domains in phospholipid monolayers.

Microscopy, Fluorescence↗
PubMed2002

[Direct fluorescence microscopy in the diagnosis of glomerulonephritis].

The use of the method of direct immunofluorescence (DIF) in the examination of the renal biopsy tissue, differentiation of various forms of glomerulonephritis, identification of immunopathogenetic mechanisms of the disease and mediators of immune reaction, are presented. In the study are described the characteristics findings of DIF in differentiation of morphologic groups of glomerulonephritis, made after light microscopy (LM). Here are presented the type and localisation of immune deposits within the kidney tissue (glomerules, blood vessels, tubules and interstitium). It was performed 52 renal biopsies in the period between 1997 and 2001 year, at the Institute of Nephrology in Sarajevo, than divided after DIF and LM in 11 different groups, with dominant membranous glomerulopathy (11 cases), diffuse mesangioproliferative with IgA nephropathy (9 cases) and minimal change glomerulonephritis (9 cases), between them. It was described the method of renal biopsy, the preservation and preparation of biopsy spacemen, and the act of fluorescence microscopy.

Complement System Proteins↗
PubMed1983

In vitro study by fluorescence microscopy and microradiography of tetracycline-tooth interaction.

The administration of tetracycline to the growing child may cause discoloration and hypoplasia in developing teeth. Whether tetracycline may also cause changes in fully mineralized enamel and dentin, is still a matter of discussion. The present investigation examines the in vitro reaction between tetracycline chloride and enamel and dentin of extracted human teeth. Specimens were immersed in 0.5 mg/ml to 20 mg/ml aqueous tetracycline solutions. After 24 hr the specimens were sectioned and studied by fluorescence microscopy and microradiography. The tetracycline solutions, which were all very acidic, were found to cause 1) demineralization of enamel and dentin, 2) incorporation of fluorescent material in enamel and, notably, dentin; and 3) formation of yellow, rhombohedral crystals on dental surfaces, especially in the more concentrated solutions. No fluorescence was seen in totally demineralized dentin. The findings indicate that the incorporation of tetracycline into enamel and dentin is caused by physiochemical processes that may take place regardless of the developmental stage of the mineralized tissues.

Dental Enamel↗
PubMed2005

Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy.

Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of deltaG++ = 44 kJ.mol(-1) and k(obs) approximately 10(5) s(-1) at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.

Animals↗
PubMed2005

The use of rhodamine 6G and fluorescence microscopy in the evaluation of phospholipid-based polymeric biomaterials.

A technique is described that allows the staining and subsequent visualization of polymers that contain the phosphorylcholine (PC) group. These materials are useful as bulk materials or coatings for the fabrication of medical devices. The staining method employs rhodamine 6G, which can be simply and rapidly applied to the polymer coating and imaged using fluorescence microscopy. The specificity of the staining for the PC polymers makes this technique suitable for the evaluation of a wide range of substrates and provides qualitative information on coating uniformity, coverage and morphology. It can be used to examine the durability of, and defects in, the coating. Statistical analysis of the fluorescent intensity by measuring the pixel value during imaging can allow for the method to be used as a quality control tool.

Biocompatible Materials↗
PubMed2005

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo.

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

Animals↗
PubMed2004

Endocytic adaptor molecules reveal an endosomal population of clathrin by total internal reflection fluorescence microscopy.

Most eukaryotes utilize a single pool of clathrin to assemble clathrin-coated transport vesicles at different intracellular locations. Coat assembly is a cyclical process. Soluble clathrin triskelia are recruited to the membrane surface by compartment-specific adaptor and/or accessory proteins. Adjacent triskelia then pack together to assemble a polyhedral lattice that progressively invaginates, budding off the membrane surface encasing a nascent transport vesicle that is quickly uncoated. Using total internal reflection fluorescence microscopy to follow clathrin dynamics close to the cell surface, we find that the majority of labeled clathrin structures are relatively static, moving vertically in and out of the evanescent field but with little lateral motion. A small minority shows rapid lateral and directed movement over micrometer distances. Adaptor proteins, including the alpha subunit of AP-2, ARH, and Dab2 are also relatively static and exhibit virtually no lateral movement. A fluorescently labeled AP-2 beta2 subunit, incorporated into both AP-2 and AP-1 adaptor complexes, exhibits both types of behavior. This suggests that the highly motile clathrin puncta may be distinct from plasma membrane-associated clathrin structures. When endocytosed cargo molecules, such as transferrin or low density lipoprotein, are followed into cells, they exhibit even more lateral motion than clathrin, and gradually concentrate in the perinuclear region, consistent with classical endosomal trafficking. Importantly, clathrin partially colocalizes with internalized transferrin, but diverges as the structures move longitudinally. Thus, highly motile clathrin structures are apparently distinct from the plasma membrane, accompany transferrin, and contain AP-1, revealing an endosomal population of clathrin structures.

Cell Line↗
PubMed2001

Combining AFM and FRET for high resolution fluorescence microscopy.

Here we demonstrate a new microscopic method that combines atomic force microscopy (AFM) with fluorescence resonance energy transfer (FRET). This method takes advantage of the strong distance dependence in Förster energy transfer between dyes with the appropriate donor/acceptor properties to couple an optical dimension with conventional AFM. This is achieved by attaching an acceptor dye to the end of an AFM tip and exciting a sample bound donor dye through far-field illumination. Energy transfer from the excited donor to the tip immobilized acceptor dye leads to emission in the red whenever there is sufficient overlap between the two dyes. Because of the highly exponential distance dependence in this process, only those dyes located at the apex of the AFM tip, nearest the sample, interact strongly. This limited and highly specific interaction provides a mechanism for obtaining fluorescence contrast with high spatial resolution. Initial results in which 400 nm resolution is obtained through this AFM/FRET imaging technique are reported. Future modifications in the probe design are discussed to further improve both the fluorescence resolution and imaging capabilities of this new technique.

Journal Article↗
PubMed2000

Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy.

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activated drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen species produced during PDT are thought to cause the destruction of tumor tissue. However, the precise mechanism of PDT is not completely understood. To provide insight into the in vitro mechanisms of PDT, we studied the subcellular localization of the photosensitizer HOSiPcOSi(CH3)2-(CH2)3N(CH3)2 (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluorescence microscopy. This technique allowed us to observe the relative distributions of Pc 4 and an organelle-specific dye within the same cell via two, spectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the fluorescence data of Pc 4 and the organelle-specific dyes were calculated. Using this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after introduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, lysosomes and Golgi apparatus, as well as other cytoplasmic membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was some loss of Pc 4 from the lysosomes as shown by the correlation coefficients. After an additional incubation period of 2 h Pc 4 slowly increased its accumulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Green FM). Both of these correlation coefficients were higher than that for Pc 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Golgi complexes.

Animals↗
PubMed2005

ELKS, a protein structurally related to the active zone-associated protein CAST, is expressed in pancreatic beta cells and functions in insulin exocytosis: interaction of ELKS with exocytotic machinery analyzed by total internal reflection fluorescence microscopy.

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitters. Here, we demonstrate the expression and function of ELKS, a protein structurally related to the CAZ protein CAST, in insulin exocytosis. The results of confocal and immunoelectron microscopic analysis showed that ELKS is present in pancreatic beta cells and is localized close to insulin granules docked on the plasma membrane-facing blood vessels. Total internal reflection fluorescence microscopy imaging in insulin-producing clonal cells revealed that the ELKS clusters are less dense and unevenly distributed than syntaxin 1 clusters, which are enriched in the plasma membrane. Most of the ELKS clusters were on the docking sites of insulin granules that were colocalized with syntaxin 1 clusters. Total internal reflection fluorescence images of single-granule motion showed that the fusion events of insulin granules mostly occurred on the ELKS cluster, where repeated fusion was sometimes observed. When the Bassoon-binding region of ELKS was introduced into the cells, the docking and fusion of insulin granules were markedly reduced. Moreover, attenuation of ELKS expression by small interfering RNA reduced the glucose-evoked insulin release. These data suggest that the CAZ-related protein ELKS functions in insulin exocytosis from pancreatic beta cells.

Animals↗
PubMed1984

Enumeration of cytoplasmic mu immunoglobulin positive acute lymphoblastic leukemia cells by flow cytometry: comparison with fluorescence microscopy.

The pre-B phenotype of childhood acute lymphoblastic leukemia, in which the mu immunoglobulin molecule is expressed in the cytoplasm of the blast cells, has been shown to have independent prognostic significance. Patients with this phenotype of the disease tend to have a poorer outcome on contemporary treatment regimens than do those with the other B-precursor phenotypes. For these clinical studies, the detection of the pre-B phenotype has depended upon fluorescent-microscope based techniques. A flow-cytometry based technique for the detection of the cytoplasmic mu immunoglobulin molecule is presented here and the results compared in detail with those of fluorescent microscopy. The results show that the two techniques are equivalent. The flow cytometry method has the advantage of a standardized control, is less labor intensive, and is observer-independent.

Cell Line↗
PubMed2000

Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant MCF-7 cells using fluorescence microscopy image analysis.

BACKGROUND: Anthracycline resistance is known to be mediated by P-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs. METHODS: The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction. Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution. Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling. RESULTS: An original image analysis procedure was developed to investigate daunorubicin and organelles co-localization. The reliability of the image analysis was controlled through chromatic shift and intensity linearity measurement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated. Although no rational relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells. CONCLUSIONS: In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular vesicles identified as Golgi vesicles and resistance to apoptosis induction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗