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At least 757 records · Page 42Linked to original sources
PubMed2005

[In vivo confocal microscopy evaluation of corneal changes induced after LASIK using the IntraLase femtosecond laser technique].

PURPOSE: To assess stromal modifications occurring after IntraLase femtosecond laser for laser in situ keratomileusis (LASIK) using the Heidelberg retina tomograph II/Rostock cornea module. MATERIAL AND METHODS: Twelve eyes from six patients were examined using the Heidelberg retina tomograph II cornea module after IntraLase femtosecond laser: ten eyes were examined at 1 week and 2 months after laser surgery, including four eyes examined at day 1, and two eyes examined at day 2. Morphological modifications of the corneal stroma, flap interface, and flap margin were evaluated at these different times and compared with the mechanical microkeratome interfaces of five patients (ten eyes), using the same technique at the same periods after the surgical intervention. RESULTS: Evaluations at days 1 and 7 showed simultaneous depletion and activation of keratocytes on both sides of the interface. We also observed some brightly reflecting particles together with scattered, less bright dots, from day 7 and increasing after 2 months. Some clinically visible deposits at the level of the interface were observed at the periphery of the flap at day 1 and could represent cell-degradation products. With confocal microscopy, they appeared as homogeneous reflective deposits with a larger size than that of particles; they had decreased at month 2. The flap margin appeared microscopically as a very clear-cut edge, including epithelial cells, while those performed with a mechanical microkeratome appeared more like a poorly limited fibrotic scar. We also observed a secondary fibrotic reaction at month 2, adjacent to the still well-defined IntraLase flap edge. CONCLUSION: This study showed substantial morphological similarity between the interfaces obtained with femtosecond laser and mechanical microkeratome. The discovery of brightly reflecting particles in the IntraLase interface goes against the hypothesis of the metallic origin of these deposits. The flap margin microscopically looked extremely well delimited, but seemed to provoke an adjacent secondary fibrotic reaction, both microscopically and macroscopically, greater at 2 months than after a mechanical microkeratome cut.

Adult↗
PubMed2002

Combined confocal microscopy and stereology: a highly efficient and unbiased approach to quantitative structural measurement in tissues.

Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure-function relationships. Conventionally structure-function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three-dimensional structure of an organ based on this two-dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three-dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in a n organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy add s enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure-function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.

Animals↗
PubMed2004

Nanoparticles, molecular biosensors, and multispectral confocal microscopy.

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.

Biocompatible Materials↗
PubMed1999

Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy.

The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

Isualization and analysis of large data collections: a case study applied to confocal microscopy data.

In this paper we propose an approach in which interactive visualization and analysis are combined with batch tools for the processing of large data collections. Large and heterogeneous data collections are difficult to analyze and pose specific problems to interactive visualization. Application of the traditional interactive processing and visualization approaches as well as batch processing encounter considerable drawbacks for such large and heterogeneous data collections due to the amount and type of data. Computing resources are not sufficient for interactive exploration of the data and automated analysis has the disadvantage that the user has only limited control and feedback on the analysis process. In our approach, an analysis procedure with features and attributes of interest for the analysis is defined interactively. This procedure is used for off-line processing of large collections of data sets. The results of the batch process along with "visual summaries" are used for further analysis. Visualization is not only used for the presentation of the result, but also as a tool to monitor the validity and quality of the operations performed during the batch process. Operations such as feature extraction and attribute calculation of the collected data sets are validated by visual inspection. This approach is illustrated by an extensive case study, in which a collection of confocal microscopy data sets is analyzed.

Algorithms↗
PubMed2005

Magnetic resonance imaging and confocal microscopy studies of magnetically labeled endothelial progenitor cells trafficking to sites of tumor angiogenesis.

UNLABELLED: AC133 cells, a subpopulation of CD34+ hematopoietic stem cells, can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected, magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma, and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However, low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature, and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors.

AC133 Antigen↗
PubMed2006

Application of 2D and 3D DIAS to motion analysis of live cells in transmission and confocal microscopy imaging.

The chemotactic signal in Dictyostelium is a cAMP wave that is relayed over relatively large distances through a cell population during aggregation. Cells exhibit unique behaviors in response to the different spatial, temporal, and concentration components of the cAMP wave, suggesting that distinct signal transduction pathways are evoked in each of the various phases of the wave. For this reason, we designed a set of experimental protocols to test responses of normal and mutant Dictyostelium amoebae to the different components of a wave of chemoattractant. We then used computer-assisted two- (2D) and three-dimensional (3D) technologies (2D and 3D Dynamic Image Analysis System [DIAS]) for analysis of cells in the absence of a chemotactic signal (basic motile behavior) and in response to the temporal, spatial, and concentration components of the wave. As a result, we have elucidated parallel and independent pathways activated by specific phases of the cAMP wave. Likewise, human polymorphonuclear neutrophils (PMNs) respond to experimentally applied waves of the chemotactic peptide fMLP, and also exhibit discrete behavioral responses to the different phases. Using Dictyostelium as a paradigm, we applied our protocols to normal and diseased human PMNs and precisely defined a chemotactic defect. In this chapter, we describe methods for quantifying behaviors in Dictyostelium amoebae, PMNs, and other amoeboid cells using 2D and 3D DIAS. These methods are useful in the reconstruction and motion analysis of most migrating cells with transmitted and/or confocal microscopy.

Animals↗
PubMed1995

Confocal microscopy of a newly identified protein associated with heart development in the Mexican axolotl.

Recessive mutant gene c for "'cardiac nonfunction" in the mexican axolotl, Ambystoma mexicanum, results in a failure of affected embryos to develop contracting hearts. Mutant embryos survive approximately 4 weeks after fertilization, but eventually die from a lack of circulation. Morphological studies show that mutant hearts lack organized sarcomeric myofibrils. This abnormality can be corrected by co-culturing early mutant hearts with normal anterior endoderm/mesoderm tissues, by culturing them in a medium "conditioned" by this normal tissue, or by RNA isolated from normal endoderm/mesoderm. Additionally, RNA isolated from normal anterior endoderm/mesoderm conditioned medium corrects the mutant hearts in a dose-dependent manner. A cDNA library is constructed using this RNA. On the basis of sequence analyses on this cDNA library, it was estimated that 56% of the total RNA present in the conditioned medium is rRNA, while 44% is nonribosomal RNA. One of the nonribosomal RNAs that showed no significant homology with other known sequences in the Genebank was examined further. An RT-PCR analysis showed that this RNA (designated "N1") is expressed in juvenile skeletal muscle, brain, and heart in significant amounts, less in the lung and not at all in the liver tissue. Affinity-purified polyclonal antipeptide antibodies were produced against the most antigenic portion of the polypeptide which was deduced from this RNA. Western blot analyses of adult heart homogenates, using these antibodies, showed a specific doublet staining at 67 kDa and 65 kDa. These doublets were purified and analyzed for their amino acid composition which showed that both bands most likely belong to the same protein. The N1-protein was further investigated to determine its localization in normal isolated hearts at embryonic stages 35, 38, and 41 and on cross-sections through the heart regions of whole normal embryos at stages 16, 33-34, 37-38, and 41-42 using immunohistochemical techniques and confocal microscopy. In addition, mutant embryos at stage 37-38 were studied for the presence and distribution of the N1-protein on cross-sections through their heart regions. The N1-protein staining was significantly reduced in mutant hearts when compared to normal.

Ambystoma mexicanum↗
PubMed2001

Confocal microscopy analysis of the activity of mitochondria contained within the 'mitochondrial cloud' during oogenesis in Xenopus laevis.

We have used ratiometric confocal microscopy and three fluorescence techniques to study the distribution and activity of mitochondria in frog oocytes during the early stages of oogenesis. Mitochondria in frog oocytes during oogenesis were characterised by a high ratio in the 'mitochondrial cloud' and perinuclear region and a low ratio in mitochondria freely dispersed within the cytoplasm. We tested whether the high ratio visualised by the three techniques represented mitochondrial membrane potential by perturbing the mitochondrial membrane potential. Carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP) caused the immediate destruction of the membrane potential, and consequent loss of fluorescence from the membrane-potential-sensitive confocal channel. In contrast, nigericin caused an increase in membrane potential represented by a steady increase in fluorescence ratio. These data demonstrate that mitochondrial activity can be measured during oogenesis in frog oocytes, and suggest that the mitochondrial cloud and perinuclear regions are characterised by highly active mitochondria.

Animals↗
PubMed1994

Chemical and physical properties of collagen implants influence their fate in vivo as evaluated by light and confocal microscopy.

Porcine collagen membranes having a rough and a smooth side were used for subcutaneous implantation studies in rats. Two tanning protocols were used for the membranes, a new one involving microwaving and glutaraldehyde treatment (NEWGA), and the other, a conventional method using glutaraldehyde treatment at room temperature (OLDGA). Untreated membranes (NONGA) were also implanted. Sections of the implants were examined by light microscopy and with the confocal laser scanning microscope focusing on neovascularization and incorporation of the implant. At 64 days, the smooth sides of NEWGA and OLDGA implants were not well incorporated, with scarring subjacent to the surface and dystrophic calcification of that side of the membrane. At the same time, the rough sides of the NEWGA and OLDGA were not calcified with a giant cell reaction around the porcine collagen. The best incorporation was found in the NONGA membranes with no dystrophic calcification, excellent neovascularization of all layers, and complete remodeling at day 64. After 5 months, the completely remodeled NONGA membrane still could be identified, and the NEWGA and OLDGA membranes were calcified with a giant cell reaction having a dense fibrous capsule. It is concluded that if cross-linking is deemed necessary, the microwave cross-linking method is advisable because in the early stages there is less reactive inflammation around it, and the implant surfaces should be rough with an open structure, making calcification of cross-linked collagen unlikely.

Cell Differentiation↗
PubMed2003

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage.

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular. In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.

Actins↗
PubMed2003

Intracellular Ca2+ measurements in live cells by rapid line scan confocal microscopy: simplified calibration methodology.

Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate CA2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3 AM loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretaton and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be used to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Aniline Compounds↗
PubMed1993

Gap junction density in human myometrium at term revealed by an anti-peptide antibody and laser scanning confocal microscopy.

The concept that the progesterone-estrogen ratio determines gap junction abundance in myometrium has developed from studies in laboratory animals. In the humans, however, gap junctions form in abundance at term despite a high progesterone-estrogen ratio. We have therefore examined the relationship between contractile activity, progesterone and estrogen levels and gap junction abundance in human myometrium at term. Myometrial tissue obtained from women who had an elective or emergency Cesarean operation at the 38th or 39th week of pregnancy was immunohistochemically examined using a polyclonal antiserum raised against a synthetic peptide matching part of the sequence of connexin43, the principal component of myometrial gap junctions. The concentration of progesterone and estradiol in the maternal blood was measured by means of radio-immunoassay. The number of gap junctions was estimated by counting of punctate staining patterns obtained in myometrial tissue as viewed by laser confocal microscopy and calculated per 1 microns 3 of tissue. The number of gap junctions measured was found to be related to the presence or absence of labor. In the myometrium of women in labor (active phase), the number of gap junctions was highest (45.25 +/- 35). When the myometrium was not contracting (non-labor) and when contractions were weak (pre-labor) the number of gap junctions was low (15.16 +/- 10-5.75 +/- 5). In the pre-and active phase of labor, the concentration of progesterone in maternal blood was significantly higher (P < 0.025 and 0.03, respectively) than in women who were not in labor. The present study shows the potential of using optical section series obtained by laser confocal microscopy for the quantification and mapping of the three-dimensional distribution of gap junctions in human myometrium at term.

Antibodies↗
PubMed1998

[High-resolution imaging of intracellular calcium ion concentration using rapid scanning confocal microscopy].

Recently, the technique for high time- and spatial-resolution imaging of intracellular calcium ion concentration ([Ca2+]i) has been developed using rapid scanning confocal microscopy to investigate calcium dynamics in restricted areas of cells. Here, the techniques are summarized and the images obtained are introduced. For such imaging, the development of fast scanning confocal microscopes was indispensable. Therefore, the UV-applicable video-rate (30 frames/sec) scanning confocal microscope in which a resonant glavanometer scanner is used for fast horizontal scans has been developed. The microscope enabled us to obtain ratio-images of [Ca2+]i using indo-1, a ratio-imaging probe. The key to success in high time- and temporal-resolution imaging is to find the best probe-loading condition, which depends on the probes and the cells. It is also very important to increase the signal-to-noise ratio value without any effects on the cells. Using the technique, we have succeeded in kinetic analysis of calcium waves in cultured hepatocytes. We have also succeeded in determining the relationship between calcium sparks to calcium transients in excitation-contraction coupling.

Animals↗
PubMedDate not supplied

Confocal microscopy in chemical neuroanatomy.

The application of computer-assisted confocal laser scanning microscopy in chemical neuroanatomy is briefly reviewed and illustrated with examples taken mainly from morphological studies of neurons in the spinal cord of the lamprey, a lower vertebrate. The principle of operation of the confocal microscopy system used, with emphasis on the computer-controlled image data acquisition and further processing of the large image volumes, is described. The imaging characteristics of confocal laser scanning microscopy in combination with fluorescence labelling, in particular the capacity to perform three-dimensional reconstructions of high optical resolution, are also described and illustrated with projection images from different viewing angles and with stereo pair reconstructions. Different possibilities for image processing, e.g. surface shading and surface extraction, are also described and illustrated.

Animals↗
PubMed2004

In vivo confocal microscopy of Thygeson's superficial punctate keratitis.

A 56-year-old Chinese man diagnosed with Thygeson's keratitis by clinical biomicroscopy was examined using a tandem scanning confocal microscope. Among normal superficial epithelial cells, clumps of markedly enlarged epithelial cells were identified. Multiple highly reflective filamentary structures were observed in the deeper layers. Most of these lesions were linear; some demonstrated curled ends and others demonstrated branching lesions with 'sprouts'. No inflammatory cells were evident in the areas of corneal stroma sampled. In vivo confocal microscopy may be helpful in the diagnosis of Thygeson's superficial keratitis. To the best of our knowledge, we report the first in vivo confocal images of focal desquamation of epithelium and intraepithelial hyper-reflective linear lesions in English literature.

Epithelial Cells↗
PubMed2005

Collagen orientation patterns in human secondary osteons, quantified in the radial direction by confocal microscopy.

The composite structure of secondary osteon lamellae, key micro-mechanical components of human bone, has intrigued researchers for the last 300 years. Scanning confocal microscopy here for the first time systematically quantifies collagen orientations by location within the lamellar thickness. Fully calcified lamellar specimens, extinct or bright in cross-section under circularly polarized light, were gently flattened, and then examined along their thickness direction, the radial direction in the previously embedding osteon. Collagen orientation was measured from confocal image stacks. So-called extinct lamellae and so-called bright lamellae are found to display distinct, characteristic patterns of collagen orientation distribution. Orientations longitudinal to the osteon axis in extinct lamellae, transverse to the osteon axis in bright lamellae, and oblique to the osteon axis in both lamellar types, show parabolic distribution through specimen thickness. Longitudinal collagen in extinct lamellae, and transverse collagen in bright lamellae, peaks at middle third of lamellar thickness, while oblique collagen peaks at outer thirds of both types. Throughout the thickness, longitudinal collagen orientations characterize extinct lamellar specimens, while orientations oblique to the original osteon axis characterize bright lamellar specimens. Measured patterns complement previous indirect results by different methods and reinforce previously hypothesized differences in lamellar mechanical functions.

Adult↗
PubMed1996

Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence.

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.

Animals↗