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PubMed2003

Immunofluorescent detection and quantitation of hepatitis A virus in sewage treatment effluent and on agri-food surfaces using scanning confocal microscopy.

An immunofluorescent (IF) assay was developed for specific detection and quantitation of hepatitis A virus (HAV) in sewage treatment effluent or attached to stainless steel, copper, high density polyethylene and polyvinyl chloride surfaces. Polyclonal antibodies were produced in rabbits and characterized for specificity to HAV. Purified anti-HAV antibodies were used in combination with ALEXA-anti-rabbit conjugate and confocal microscopy for detection and quantitation of HAV in effluent samples. Using this immunological approach, as little as 2 x 10(5) PFU/ml were detected and the signal generated was proportional to the concentration of HAV up to 2 x 10(8) PFU/ml. Counts obtained by IF assay were highly correlated with those of the SYBR Green II nucleic acid labelling method (r(2)=0.995) and conventional plaque assay (r(2)=0.988). The IF assay described is rapid (3 h), sensitive, specific to HAV and suitable for qualitative as well as quantitative studies.

Animals↗
PubMed1996

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies.

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live and in situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.

Antibiotics, Antineoplastic↗
PubMed1995

Chemical and immunological heterogeneity of fibrillar amyloid in plaques of Alzheimer's disease and Down's syndrome brains revealed by confocal microscopy.

Amyloid beta peptides (A beta) are deposited in the brains of Alzheimer's disease (AD) and elderly Down's syndrome (DS) patients in a variety of amyloid plaques. Among these are classical plaques composed of a spherical core and corona. Analyzing AD tissue sections single and double stained with anti-A beta antibodies and thioflavin S (thioS) by bright field, fluorescence, and confocal microscopy revealed that spherical plaque cores consist of a thioS-positive center and an anti-A beta antibody immunoreactive rim. This indicates that there is a fibrillar form of amyloid that is thioS positive but not immunoreactive with anti-A beta antibodies. In contrast, classical plaques in DS patients have irregular cores that are thioS positive as well as anti-A beta immunoreactive. In addition, a subset of plaques in both DS and AD patients have a distinct "fibrous" appearance when stained with thioS, but are amorphous when immunostained. These findings suggest that anti-A beta antibodies and thioS stain similar; as well as different forms of fibrillar amyloid. A beta may become thioS positive by interacting with one or more of its known molecular chaperons, and this may be important for the pathogenesis of AD, given that thioS-positive A beta deposits are associated with neuritic and synaptic damage.

Aged↗
PubMed2005

Evaluation of human knee meniscus biopsies with near-infrared, reflectance confocal microscopy. A pilot study.

Knee cartilage biopsy is used to confirm the pathology in both clinical and experimental conditions and often guides diagnosis and therapeutic strategies. Current histopathological techniques are time consuming, induce tissue artefacts and often prevent further evaluation, once the tissue has been fixed. Hence, there is a potential need for a fast and nondestructive imaging technique for unfixed tissue. Near-infrared, reflectance confocal microscopy (CM) allows real-time, virtual sectioning of unstained, bulk tissue samples. This pilot study evaluates the use of CM in the assessment of meniscus histopathology in a series of 26 freshly-excised human meniscus samples compared to standard light microscopy of stained sections. CM images of the meniscus show cell and matrix detail, depicting morphologic features of collagen and elastic fibres, vessels and nerve endings. In addition, crystal deposits of gout and pseudogout are also demonstrable. Thus, CM is a novel imaging technique that could enable the pathologist to make a rapid microscopic evaluation of cartilage in a fresh and unfixed fashion.

Adult↗
PubMed2001

4Pi-confocal microscopy of live cells.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

Escherichia coli↗
PubMed2006

Three-dimensional visualization of microvessel architecture of whole-mount tissue by confocal microscopy.

The three-dimensional architecture of the nascent microvascular network is a critical determinant of vascular perfusion in the setting of regenerative growth, vasculopathies and cancer. Current methods for microvessel visualization are limited by insufficient penetration and instability of endothelial immunolabels, inadequate vascular perfusion by the high-viscosity polymers used for vascular casting, and destruction of tissue stroma during the processing required for scanning electron microscopy. The aim of this study was to develop whole-mount tissue processing methods for 3D in situ visualization of the microvasculature that were also compatible with supplementary labeling for other structures of interest in the tissue microenvironment. Here, we present techniques that allow imaging of the microvasculature by confocal microscopy, to depths of up to 1500 mum below the specimen surface. Our approach includes labeling luminal surfaces of endothelial cells by i.v. injection of fluorescently conjugated lectin and filling the microvasculature with carbon or fluorescent nanoparticles/Mercox, followed by optical clearing of thick tissue sections to reduce light scatter and permit 3D visualization of microvessel morphology deep into the sample. Notably, tissue stroma is preserved, allowing simultaneous labeling of other structures by immunohistochemistry or nuclear dyes. Results are presented for various murine tissues including fat, muscle, heart and brain under conditions of normal health, as well as in the setting of a glioma model growing in the subcutaneous space or orthotopically in the brain parenchyma.

Animals↗
PubMed1996

Perimetric [Ca2+]i rise and exocytosis detected by ultraviolet laser scanning confocal microscopy in rat peritoneal mast cells.

It has been reported that in dialysed mast cells an increase in mean [Ca2+]i is neither necessary nor sufficient for secretion; however, it is possible that juxtamembranal [Ca2+]i may exceed the mean [Ca2+]i before exocytosis. The present study was carried out to analyse spatial and temporal dynamics of [Ca2+]i and concomitant exocytosis in intact rat peritoneal mast cells using UV laser scanning confocal microscopy. Stimulation with Compound 48/80 (16 microM) increased mean [Ca2+]i, causing an initial rapid elevation from 40 to 200 nM, which lasted for 6 s and was followed by a delayed secondary increase to 600 nM. Exocytotic images were seen in the cell perimeter 16 s after the stimulation. Perimetric and nuclear [Ca2+]i increased to several mumoles per litre, while that in the intermediate region remained low. In a Ca(2+)-deficient environment, Compound 48/80 still increased perimetric [Ca2+]i to micromolar values and induced exocytosis. This study clearly indicates, for the first time, that a perimetric increase in [Ca2+]i to micromolar levels precedes exocytosis, in intact, as opposed to permeabilized cells.

Animals↗
PubMedDate not supplied

In vivo confocal microscopy in the acute phase of corneal inflammation.

Two cases of noninfectious keratitis were studied using in vivo corneal confocal microscopy in addition to routine slit-lamp biomicroscopy. A 10-year-old boy suffered from keratitis in his left eye due to a bee sting and a 20-year-old man had keratitis following corneal blunt trauma. In both cases, we found a honeycomb pattern at the anterior and mid-stromal level of the middle cornea. This honeycomb pattern disappeared in 1 week with steroid treatment. This pattern might be caused by syncytial cell bodies of activated keratocytes, by focal corneal stroma edema, or by polymorphonuclear leukocyte infiltration along with the keratocytes or along preformed channels within the corneal stroma known as Bowman channels. Further analysis in a large number of patients may aid in further understanding in vivo human corneal inflammation.

Acute Disease↗
PubMed2005

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros.

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.

Animals↗
PubMed2001

Measurement of corneal sublayer thickness and transparency in transgenic mice with altered corneal clarity using in vivo confocal microscopy.

Measurement of sublayer thickness and transparency at cellular level in the living animal are critical to understanding the role of specific transgenes and transgene products in controlling corneal development and maintenance of transparency. Using two different transgenic mouse strains having altered corneal clarity, we have evaluated the ability of in vivo confocal microscopy to measure corneal haze and localize light scattering structures. Projection of 2-D and 3-D image information identified the nature and location of light scattering within the cornea and allowed correlation of unique structural differences to transgene expression. Our findings suggest that in vivo confocal microscopy can be used to identify the effects of transgene expression on mouse corneal transparency.

Animals↗
PubMed1998

4D confocal microscopy of Dictyostelium discoideum morphogenesis and its presentation on the Internet.

Methods to present three-dimensional (3D) and time series of 3D datasets (4D) are demonstrated using the recent advances in confocal microscopy and computer visualization. The process of cell sorting during tip formation in the slime mould Dictyostelium discoideum is examined as an example by in vivo confocal microscopy of spectrally different green fluorescent protein (GFP) variants as reporters of cell-type specific gene expression. Also, cell sorting of the co-aggregating slime mould species D. discoideum and D. mucoroides is observed using a GFP variant and a spectrally distinguishable fluorescent vital stain. The confocal data are handled as 3D and 4D datasets, their processing and the advantages of different methods of visualization are discussed step by step. Selected sequences of the experiments can be viewed on the Internet, giving a much better impression of the complex cellular movements during Dictyostelium morphogenesis than printed photographs.

Animals↗
PubMed2006

Characterization of Acanthamoeba-microsphere association by multiparameter flow cytometry and confocal microscopy.

BACKGROUND: Acanthamoebae, in common with other protozoa, readily endocytose particulate material, which in turn may lead to the spread of infectious disease. METHODS: Evaluation and quantification of plain and carboxylate FITC-microsphere association with acanthamoebal trophzoites was undertaken using a combination of flow cytometry and confocal microscopy. Trophozoites from strains and species of Acanthamoeba were exposed to plain and carboxylate FITC-microspheres. Microsphere size and aspects such as trophozoite starvation, maturity, and exposure to metabolic inhibitors were assessed. RESULTS: All species and strains of Acanthamoeba readily endocytosed plain and carboxylate microspheres. Starving trophozoites significantly increased binding and potential ingestion of microspheres, whereas trophozoites of increasing maturity lost such abilities. Trophozoites showed a significant preference for 2.0- and 3.0-microm-diameter microspheres when compared with other sizes, which in turn could occupy much of the cytoplasm. The physiological inhibitors sodium azide, 2,4-dinitrophenol, and cytochalasin B reduced microsphere association with trophozoites; however, some microspheres still bound and associated with trophozoites after inhibitor exposure, a manifestation of both active and inactive agent involvement in microsphere endocytosis. CONCLUSIONS: Even though the origins of microsphere binding by acanthamoebal trophozoite remains shrouded, the combination of flow cytometry and confocal microscopy supported synergistic quantification and qualification of trophozoite-microsphere endocytosis.

Acanthamoeba↗
PubMedDate not supplied

Extent of the odontoblastic process. Analysis by SEM and confocal microscopy.

BACKGROUND: Towards the middle of the eighteenth century, Tomes described membranous structures of cellular origin inside the dentinal tubules. Subsequent studies of the terminal segment of the odontoblasts have been controversial. According to Fusayama, this cellular process reaches as far as the junction; others, including Brännström, affirm that this process is present only in the inner third of the dentin. AIMS: The aim of the present study was to verify the exact length of the odontoblastic terminal segment with the aid of advanced confocal microscopy (CLSM) and high resolution field-emission scanning electron microscopy (FE-SEM). METHODS: Premolars due for extraction for orthodontic reasons, were used for this study; vestibular class V cavities about 2 mm in diameter and 3 mm in depth prepared and subsequently filled with IRM (Caulk Dentsply). All teeth were extracted after four weeks fixed and prepared for examination under SEM and CLSM. RESULTS: CLSM revealed cylindrical structures inside the tubules even in the absence of odontoblasts. SEM evidenced the presence tubular structures only in the inner third of the dentin (towards the pulp). CONCLUSIONS: Structures previously described as cellular processes can be identified as the lamina limitans of the peritubular dentin.

Bicuspid↗
PubMed2005

Dynamic regulation of Tec kinase localization in membrane-proximal vesicles of a T cell clone revealed by total internal reflection fluorescence and confocal microscopy.

Tec family tyrosine kinases are key regulators of lymphocyte activation and effector function. Several Tec family kinases (Tec, Itk, Rlk/Txk) are expressed in T cells, but it is still not clear to what degree these are redundant or have unique functions. We recently demonstrated that Tec alone, among the Tec kinase family members examined, can induce nuclear factor of activated T cell-dependent transcription. This unique functional characteristic correlated with a unique pattern of subcellular localization, as Tec (but not other family members) was found in small vesicles, the appearance of which requires signaling through the T cell receptor for antigen. Here we report on our studies of these Tec-containing structures in live T cells, using total internal reflection fluorescence microscopy. With this technique, we showed that, in live T cells, the Tec vesicles are located at the plasma membrane, the vesicles are unique to Tec (and not the related kinase Itk), and their formation and maintenance require T cell receptor signaling through Src family kinases and PI 3-kinase. Finally, we have imaged isolated T cell membranes by confocal microscopy, confirming the membrane-proximal location of Tec vesicles, as well as demonstrating overlap of these vesicles with the tyrosine kinase Lck, the Tec substrate PLC-gamma1, and the early endosomal antigen 1 marker EEA1.

Animals↗
PubMed2005

Visualisation of morphological changes in living intact human microvessels using confocal microscopy.

Conventional techniques to visualise microvascular structure often involve fixed tissue slices that provide two-dimensional images. A previous study using diffusive labelling of fresh, dissected tissue samples with fluorescently-tagged endothelial markers demonstrated the possibility of examining the three-dimensional architecture of the microvasculature using confocal microscopy. The present study extends the use of this quick and simple method of diffusive labelling to examine the possibility of repeatedly measuring changes in the morphology of intact microvessel in response to pharmacological stimuli. Initially, three-dimensional surface-rendered images of the same microvessel derived from the placenta and subcutaneous biopsies demonstrated morphological and topological changes in response to temperature and increasing potassium changes of physiological salt solutions, respectively. Furthermore, a dose-response study was performed with subcutaneous microvessels using the potent vasodilator, adrenomedullin. Analysis of a series of z-stack, superimposed to form a single maximum brightness image, demonstrated an inverse dose-response relationship, with responses to increasing adrenomedullin concentrations (10(-12) to 10(-8) M). In vessels that had constricted in response to noradrenaline (diameters: 22.4 to 58.0 microm), physiological concentrations of 10(-12) M increased vessel diameter by 108% above baseline conditions. Control treatment using physiological salt solution did not demonstrate any changes. The technique described suggest that diffusive labelling with vascular endothelial markers such as ulex europeaus agglutinin I in live tissue samples may be used in conjunction with confocal microscopy to demonstrate heterogeneous morphological and topological changes in intact segments of the microvasculature.

Adrenomedullin↗
PubMed2006

Intense pulsed light therapy for superficial pigmented lesions evaluated by reflectance-mode confocal microscopy and optical coherence tomography.

Intense pulsed light (IPL) therapy is reported to be effective for pigment removal from pigmented lesions. However, the dynamic mechanism of pigment removal by IPL therapy is not completely understood. We investigated the mechanism of IPL therapy for the removal of pigmented skin lesions through non-invasive observation of the epidermis. Subjects with solar lentigines on the face were treated with three sessions of IPL therapy. The solar lentigines were observed on consecutive days after the treatments using reflectance-mode confocal microscopy (RCM) and optical coherence tomography (OCT). In addition, desquamated microcrusts that formed after the treatment were investigated by transmission electron microscopy (TEM). The images of RCM and OCT showed that the melanosomes in the epidermal basal layer rapidly migrated to the skin surface. The TEM images of the extruded microcrusts revealed numerous melanosomes together with cell debris. It was also found that the IPL irradiated melanocytes in the lesions seemed to be left intact and resumed their high activity after treatment. We conclude that IPL therapy effectively removed the dense melanosomes in the epidermal-basal layer. However, additional application of suppressive drugs such as hydroquinone or Q-switched laser irradiation is necessary to suppress the remaining active melanocytes.

Humans↗
PubMed2005

A microbiological and confocal microscopy study documenting a slime-producing Staphylococcus epidermidis isolated from a nylon corneal suture of a patient with antibiotic-resistant endophthalmitis.

BACKGROUND: We describe a case of posttraumatic endophthalmitis unresponsive to systemic (amoxicillin+clavulanic acid and piperacillin/tazobactam), intra-ocular (vancomycin) and topical (ofloxacin, tetracycline and sulfametoxazole) antibiotic therapy. Microbiological and confocal microscopy studies of a nylon corneal suture revealed the presence of a slime-producing strain of Staphylococcus epidermidis. METHODS: We describe the history and clinical presentation of a 77-year-old man in whom a high-grade posttraumatic endophthalmitis resolved only after the removal of a single nylon corneal suture. Microbiological investigations of the aqueous, vitreous and suture were performed, and the propensity of the suture-associated isolate to form biofilm was assessed using confocal microscopy. RESULTS: A single stain of S. epidermidis was isolated from both aqueous and vitreous specimens and from the suture. The planktonic form of the isolate was susceptible in vitro to the antibiotics administered to the patient, but the strain was capable of forming biofilms and this phenotype showed resistance to high concentrations of the same antibiotics. CONCLUSIONS: The presence of a slime-producing strain of S. epidermidis should be considered in endophthalmitis that is unresponsive to specific antibiotic therapy, especially in cases in which an intra-ocular foreign body (e.g., a suture) is present.

Aged↗
PubMed2003

Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin.

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.

Arm↗