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PubMed2006

Plastic-to-elastic transition in aggregated emulsion networks, studied with atomic force microscopy-confocal scanning laser microscopy microrheology.

In this paper, we demonstrate how the simultaneous application of atomic force microscopy (AFM) and confocal scanning laser microscopy (CSLM) can be used to characterize the (local) rheological properties of soft condensed matter at micrometer length scales. Measurement of AFM force curves as a function of the indentation amplitude and speed (magnitude and direction) can produce a "mechanical fingerprint" that contains information about material stiffness, hysteretic losses, and time scales for stress relaxation and/or network recovery. The simultaneous CSLM visualization of changes in the material's structure provides complementary information about how the material accommodates the indentation load. Since these experiments are done on areas of O(100 microm2) on materials having a surface of O(1 cm2), the measurements can be repeated on "fresh" material many times, contrary to traditional rheometers where the whole sample is loaded at once. As a particular example, we consider the case of a network of aggregated water-in-oil (W/O) emulsion droplets, in which the mechanical behavior changes drastically over time. Whereas the freshly prepared material shows a soft plastic behavior, after a time lapse of several weeks, the very same sample shows a much stiffer and elastic response. This drastic change in behavior is clearly reflected both in the signature of the AFM force curves and in (the reversibility of) the structural deformations observed with CSLM. The fact that these drastic mechanical changes take place without significant changes in the structure of the material (before loading) indicates that the stiffening of the droplet network is caused by an increase in the strength of the bonds between droplets. A remarkable finding for the elastic droplet network is that, while the structure recovers completely after the indenter is taken out, there is still an appreciable hysteresis in the force curves, indicating that dissipation also occurs. This hysteresis was not found to depend on the indentation speed.

Journal Article↗
PubMed2003

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

Anestrus↗
PubMed1994

Cornea: confocal microscopy.

The confocal microscope opens a whole new window in early diagnosis of ocular conditions. Previously, details at the cellular level could only be viewed with conventional microscopes in a laboratory setting. By using confocal microscopy, results can now be obtained instantaneously in the living human eye. This non-invasive high magnification technique provides real-time images of cornea morphology.

Cornea↗
PubMed1992

From two dimensional (2D) to three dimensional (3D) analysis by confocal microscopy.

The confocal microscope is becoming increasingly important as an apparatus to analyze the 3-D topography of the cell. Main reasons are the high resolution optical sectioning capacity, the non-invasiveness which leaves the object intact, and the imaging capabilities. This chapter introduces a description of the confocal principle, the basic concepts of confocal fluorescence microscopy and some criteria for cell preservation. Optimization of in situ immunofluorescence, hybridization and detection procedures in combination with new digital microscope techniques can fully express their capacities only if the preparation of biological specimens is accurate for 3-D analysis. Some applications of confocal microscopy to the study of intranucleolar antigens, enzyme translocations and fluorescence in situ hybridization, are described in association with 3-D software image processing, as a useful framework for the study of the 3-D visualization of proteins and chromatin domains.

Histocytological Preparation Techniques↗
PubMed1994

Preparation of chromosome spreads for electron (TEM, SEM, STEM), light and confocal microscopy.

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactions.

Chromosomes, Human↗
PubMed2002

In vivo examination of lentigo maligna and malignant melanoma in situ, lentigo maligna type by near-infrared reflectance confocal microscopy: comparison of in vivo confocal images with histologic sections.

In vivo confocal microscopy can noninvasively image thin en face sections within living intact human tissue with high resolution and contrast. This evolving technique may provide clinicians with tools to help detect lentigo maligna lesion progression in vivo and may be important in defining tumor margins, thus providing a more definitive surgical eradication of lentigo maligna and malignant melanoma in situ, lentigo maligna type. We present a case of malignant melanoma in situ, lentigo maligna type, and we describe the images seen with confocal microscopy in correlation with routine histopathology.

Aged↗
PubMed2006

Diagnosis of epithelial ingrowth after penetrating keratoplasty with confocal microscopy.

PURPOSE: To report confocal microscopy use in the clinical diagnosis of epithelial ingrowth after penetrating keratoplasty (PKP). METHODS: A 36-year-old female patient with keratoconus developed a well-delimited posterior hazy membrane covering the inferior two thirds of the cornea 3 months after an uneventful PKP. A posterior corneal line was present resembling an endothelial graft rejection line, but with no keratic precipitates or corneal edema. Ocular hypertension was not observed. Confocal microscopy was performed to elucidate the diagnosis. RESULTS: Confocal microscopy showed epithelium and stroma with normal findings. Two distinct cellular types were presented at the endothelium layer. Enlarged endothelial cells were observed in the superior part of the cornea up to the leading edge of the hazy membrane. In the middle and inferior part of the graft, the cells were larger, with polygonal shape and easily recognizable hyperreflective nuclei, suggestive of epithelial cells. With these confocal microscopy findings, the patient was promptly submitted to another PKP. Histologic analysis confirmed the diagnosis of epithelial ingrowth. CONCLUSION: Confocal microscopy imaging technique seems to be a useful tool in the early diagnosis of epithelial ingrowth after PKP.

Adult↗
PubMed2005

In vivo assessment of melanocytic nests in nevi and melanomas by reflectance confocal microscopy.

In vivo reflectance confocal microscopy is a novel technique for the noninvasive study and diagnosis of the skin. The aim of this study was to describe and characterize the cytological and architectural aspects of cell clusters in melanocytic lesions observed by confocal microscopy, and to correlate them with routine histopathology. A total of 55 melanocytic lesions comprising 20 melanomas, 25 acquired nevi and 10 Spitz nevi were studied by means of reflectance confocal microscopy, dermoscopy and routine histopathology. Three different types of cell clusters at confocal microscopy observation (dense, sparse cell and cerebriform clusters) were identified and correlated with histopathology. Dense clusters appeared characteristic for benign lesions, although present in 13 out of 20 melanomas. Sparse cell clusters were more frequently observable in melanomas, but also sporadically present in one Spitz nevus. Moreover, cerebriform clusters were exclusively observed in five out of 20 melanomas. Confocal microscopy allowed the in vivo characterization of aspects of melanocytic nests and their exact correlation with histopathology.

Humans↗
PubMed1999

Confocal microscopy in biomedical research.

Confocal microscopy has allowed a major advance in biological imaging, since it represents a rapid, cost effective means of ecamining thick tissue specimens. In most cases, this involves fluorescence imaging and it is increasingly being used as a basic tool in biomedical research. Confocal microscopy allows the collection of thin optical sections, without the need for physical sectioning of the tissue. Additionally, confocal microscopes can usually produce images with greater sensitivity, contrast and resolution than those produced with normal light microscopes. We attempt to explain how this technology might be better used as a routine research tool. Since high quality, in-focus optical sections of thick tissue preparations can be generated quickly, confocal microscopy, in combination with immunofluorescence histochemistry, can now be used to examine complex three-dimensional distributions of distinct structures within tissues such as nerves within airways. Additionally, ultraviolet confocal microscopy allows the assessment of both dynamic and static phenomena in living cells and tissues. Thus, in addition to the imaging of fluorescence associated with structural elements, confocal microscopes can be used to quantitatively evaluate the distribution and fluxes of intracellular ions like calcium. Rapid, line-scanning confocal microscopes can be used in the assessment of dynamic events. For example, the in vivo imaging of microvascular permeability in airways becomes possible for the first time. By providing examples of some of our uses for confocal microscopy, we might encourage others to explore this relatively new and important texhnology for examining events and structures in single cells, tissue samples and in intact animals.

Animals↗
PubMed1991

Laser scanning and confocal microscopy of daunorubicin, doxorubicin, and rhodamine 123 in multidrug-resistant cells.

The multidrug-resistant gene (MDR1) encodes an energy-dependent drug efflux pump (P-glycoprotein) for many anti-cancer drugs. We have studied the intracellular distribution of rhodamine 123 (R123), daunorubicin (DN), and doxorubicin (DOX) in cells expressing a human MDR1 gene. The distribution of these fluorescent drugs was measured by laser scanning microscopy and confocal microscopy. We devised a new method for analysis of fluorescence line scan data to determine the intracellular distribution of fluorescent probes. This method and confocal microscopy showed that R123, DN, and DOX are localized to both plasma membrane and intracellular compartments in multidrug-resistant cells. When the cells are treated with verapamil, an inhibitor of the multidrug transporter, the amount of DOX, DN, and R123 associated with the cell rises. After inhibition, the relative distribution of DOX and DN between the cell surface and intracellular structures does not change dramatically. However, R123 tends to relocalize to intracellular sites from predominantly plasma membrane sites, indicating that this dye behaves differently than the anti-cancer drugs. These results show the subcellular distributions of R123, DN, and DOX in plasma membrane, cytoplasm, and intracellular membrane systems, but do not allow definitive distinctions among existing models of how P-glycoprotein affects the distribution of drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Theoretical basis of confocal microscopy.

A confocal microscope forms its image by recording light primarily from a small focal volume, largely ignoring points to the side or above or below. That volume, described as a point-spread function, is the product of two similar functions that are generated by the objective lens. Because of that multiplication, the recorded light is greater than even the integrated total of the light from all other points in a thick sample. Some of the implications of implementing this theory are reflected in the choices available to users of confocal microscopes.

Fluorescence↗
PubMed2001

In vivo confocal microscopy in dermatology.

Confocal microscopy is an optical imaging tool that allows for high resolution, noninvasive imaging in vivo. Thin sections of human tissue can be imaged allowing visualization of cellular and nuclear detail without biopsy. This technique recently has been used to image benign and malignant pigmented skin lesions, nonmelanoma skin cancer, inflammatory skin conditions, and dynamic skin processes.

Dermatology↗
PubMed1998

Visualization and quantitation of iontophoretic pathways using confocal microscopy.

Laser scanning confocal microscopy (LSCM) has been used to visualize and quantitate the penetration of a model, anionic, fluorescent compound (calcein) along the iontophoretic transport pathways within hairless mouse skin. The LSCM technique permits optical sectioning of full-thickness, unfixed tissue, thereby avoiding poor image quality due to blurring from out-of-focus fluorescence, and obviating artifactual redistribution of the permeant during processing. Simple measurements of the approximate flux of the probe across hairless mouse skin were also made using standard in vitro diffusion cell methodology and a fluorometric assay. Most importantly, LSCM imaging strategies were developed to overcome depth-dependent sensitivity problems. These visualization studies showed that iontophoresis of calcein into hairless mouse skin enhanced delivery, particularly via follicular structures, to significant depths into the barrier. Nonfollicular transport was also apparent, especially at more superficial levels. Quantitative analyses of the LSCM images showed that, although significant nonfollicular transport occurs, the efficiency of the follicular pathway, when the relative surface area is taken into account, is considerable. Overall, therefore, this work contributes significantly to the ultimate goal of fully comprehending the mechanism(s) of iontophoretic drug delivery across the skin.

Animals↗
PubMed1996

A KATP channel opener protects cardiomyocytes from Ca2+ waves: a laser confocal microscopy study.

Laser confocal microscopy was used to visualize intracellular spatiotemporal Ca2+ patterns in single guinea pig ventricular myocytes loaded with the Ca2+ indicator, fluo 3-acetoxymethyl ester (fluo 3-AM), and exposed to moderately elevated extracellular K+ to induce partial membrane depolarization. Analysis of K(+)-induced intracellular Ca2+ elevation revealed three distinct paradigms: 1) diffuse, nonoscillatory Ca2+ elevation across the myocyte; 2) localized Ca2+ elevation in anatomically restricted areas (Ca2+ sparks); and 3) regenerative frontal propagations of Ca2+ that traversed the length of the cell (Ca2+ waves). The first two patterns were more frequently observed when the extracellular K+ concentration was raised to 8 mM. Ca2+ waves became more common when extracellular K+ concentration was increased to 16 mM, suggesting that a minimum threshold of increase in intracellular Ca2+ is necessary for the organization of Ca2+ waves. The velocity of propagation was typically approximately 60 microns/s with an average frequency of one wave per second crossing at a given point in the cell. Wave propagation resulted in spatial and temporal oscillations in cytosolic and nuclear Ca2+ concentration. Treating cardiac cells with aprikalim, a potassium channel-opening drug, prevented 16 mM K+ (but not 32 mM K+) from inducing an increase in Ca2+ concentration and from generating Ca2+ waves. In cardiomyocytes treated with glyburide, a selective antagonist of ATP-sensitive K+ channels, aprikalim failed to prevent 16 mM K+ from inducing Ca2+ waves. In summary, moderate hyperkalemia induces distinct nonuniform form patterns of intracellular Ca2+ elevation in ventricular cells, which can be prevented by a potassium channel-opening drug through a glyburide-sensitive mechanism.

Adenosine Triphosphate↗
PubMed1997

Rapid coupling of calcium release to depolarization in Limulus polyphemus ventral photoreceptors as revealed by microphotolysis and confocal microscopy.

Microphotolysis and confocal microscopy were used to investigate the timing of calcium release and of the electrical response in Limulus polyphemus ventral photoreceptors. The fluorescent dyes Fluo-3 and Calcium Green-5N were used to monitor local Ca2+ elevations. Photolysis of caged inositol trisphosphate (InsP3) close to the plasma membrane of the light-sensitive rhabdomeral (R-) lobe resulted in Ca2+ elevation within 10-20 msec, 20-45 msec before the physiological response to light normally would be detected. Inward ionic current flow and depolarization followed InsP3-induced calcium release within 2.5 +/- 3.3 msec. Voltage-clamping the cells and removal of extracellular Ca2+ did not affect the timing of the Ca2+ elevation that followed the photolysis of caged InsP3 or its relationship to the electrical response. In contrast to the physiological response to light, which only released calcium within the R-lobe, photolysis of InsP3 elevated Cai in both lobes, although with much greater effect in the R-lobe, as compared with the bulk of the A-lobe, suggesting the presence of InsP3-sensitive calcium stores in both lobes. Photolysis of caged calcium [o-nitrophenyl EGTA (NPE)] at the edge of the R-lobe activated an inward ionic current within 1.8 +/- 0.7 msec. This NPE-induced current reversed at a membrane potential of 10 +/- 6 mV in the range typical of that of the light-activated current under physiological conditions. Calcium release, therefore, activates an inward current rapidly enough to contribute to the electrical response to light.

Action Potentials↗
PubMed1995

Light activated calcium release in Limulus ventral photoreceptors as revealed by laser confocal microscopy.

Using confocal imaging and fluorescent calcium indicators, light-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) in Limulus ventral photoreceptors was shown to be initiated within 4 microns of the light-sensitive plasma membrane. Within 500 ms, elevation of [Ca2+]i spread throughout the light-sensitive rhabdomeral lobe of the photoreceptor, but barely penetrated the arhabdomeral lobe. During saturating illumination of measurement spots near the plasma membrane, [Ca2+]i rose at rates of 1-2 mM/s after a latent period of 14-40 ms, reaching peak concentrations of approximately 150 microM. Rapid elevation of [Ca2+]i persisted in the absence of extracellular Ca2+ and was therefore ascribed to release from intracellular stores. The elevation of [Ca2+]i was always detectable within 5 ms of the electrical response of the photoreceptor to light. In 14 out of 54 measurements, detection of elevated calcium preceded the electrical response. Cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca-pumps, greatly reduced the elevation of [Ca2+]i during bright flashes and the sensitivity of the electrical response to dim flashes. However, the maximal response to bright flashes was not diminished. Therefore, although the calcium release that we detect may be fast enough to contribute to the electrical response we are unable to demonstrate that it is absolutely required.

Animals↗
PubMed1997

Redistribution of DNA topoisomerase II beta after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy.

Using confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase II beta (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37 degrees or 42 degrees C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0 degree C in a buffer containing spermine/spermidine/KCl or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0 degree C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37 degrees or 42 degrees C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KCl, the incubation buffer also contained 5 mM Mg++ and the temperature was 37 degrees C. If the stabilization was performed at 42 degrees C, Mg++ failed to maintain the original distribution of DNA topoisomerase II beta, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37 degrees C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37 degrees C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0 degree C incubated nuclei; they showed a certain degree of shrinkage in 0 degree C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37 degrees or 42 degrees C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37 degrees C, but not at 42 degrees C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus.

Antibodies, Monoclonal↗
PubMed2005

Endoscopic confocal microscopy.

PURPOSE OF REVIEW: Endoscopic confocal microscopy is a new endoscopic imaging technology that produces high-magnification cross-sectional images of the gastrointestinal epithelium during endoscopy. These high-magnification images might allow the endoscopist to make a tissue diagnosis during endoscopy without biopsy and histopathology. The purpose of this article is to review the salient features of endoscopic confocal microscopy, describe current clinical research areas using the technology, and depict its potential role in the management of gastrointestinal diseases. This review includes an overview of the technology, a review of recent publications describing its function, and a discussion of potential applications. RECENT FINDINGS: Endoscopic confocal microscopy has been technologically feasible only for a short time; therefore, the published experience is limited. Studies describing the use of endoscopic confocal microscopy for the detection of dysplastic tissue have been limited to a single paper in which endoscopic confocal microscopy was reported to be highly sensitive and specific for the detection of colonic dysplasia in a general screening population. Active areas of investigation include detecting dysplasia in ulcerative colitis and Barrett's esophagus. A recent case report detailed the ability of endoscopic confocal microscopy to detect gastric Helicobacter pylori in vivo. SUMMARY: Endoscopic confocal microscopy is a new imaging technology that produces high-magnification cross-sectional images of the gastrointestinal tract. Its role in detecting and managing diseases of the gastrointestinal tract has been studied in colon cancer screening and is currently being evaluated in a variety of premalignant conditions.

Endoscopes, Gastrointestinal↗