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PubMedDate not supplied

Wide field scanning slit in vivo confocal microscopy of flattening-induced corneal bands and ridges.

We used a wide field scanning slit confocal microscope to examine the response of the in vivo human cornea to flattening. Flattening-induced effects consisted of (1) anterior corneal mosaic, which appeared as a meshwork of intersecting stromal and Bowman's layer bands with overlying epithelial ridges; (2) deep and middle stromal bands, which were narrower than and unrelated in position to the anterior corneal mosaic; and (3) posterior surface ridges. The posterior surface ridges projected posteriorly into the anterior chamber consisted of endothelium, Descemet's membrane, and posterior stroma, and were unrelated in position to posterior stromal bands. Confocal microscopy is a promising modality in the examination of the cornea and its response to mechanical stress.

Cornea↗
PubMed2004

Confocal microscopy evaluation of stromal ablation depth after myopic laser in situ keratomileusis and photorefractive keratectomy.

PURPOSE: To compare the measured ablation depth after myopic laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) with the predicted ablation depth. SETTING: Mayo Clinic, Rochester, Minnesota, USA. METHODS: Twenty-five eyes of 15 patients had PRK and 25 eyes of 15 patients had LASIK to correct refractive errors between -1.50 diopters (D) and -11.00 D. The corneas were examined by in vivo confocal microscopy before and 1 month after both procedures. Thickness measurements were obtained from digital-image analysis of confocal scans. The measured ablation depth, an estimate of the actual photoablation depth, was obtained as the surgically induced stromal thinning between the preoperative and the 1-month post-PRK or post-LASIK central stromal thickness. The predicted ablation depth was recorded from the laser's software program. RESULTS: In LASIK, the measured ablation depth (81 microm +/- 34 [SD]) was 25% greater than the predicted ablation depth (65 +/- 13 microm, P =.007) and the difference between the measured and predicted ablation depths was positively associated with the mean ablation depth (r = 0.81, P<.001). In PRK, there was no difference between the measured ablation depth (48 +/- 19 microm) and the predicted ablation depth (47 +/- 18 microm, P =.84). CONCLUSION: Significantly more tissue than predicted was removed by LASIK than by PRK excimer photoablation with the laser system used in this study.

Adult↗
PubMedDate not supplied

Changing paradigms in dermatology: confocal microscopy in clinical and surgical dermatology.

The current practice of pathology and dermatopathology depends upon the evaluation of tissue in some manner extirpated from the patient and then processed and stained. While high resolution of detail can be accomplished by this method, there are certain risks and disadvantages. Recent imaging techniques now allow for a potential of achieving noninvasive high-resolution analysis of lesions in situ in the patient. Of these, Reflectance mode confocal microscopy offers the highest resolution imaging comparable to routine histology. Being entirely non invasive, skin can be observed in its native, dynamic state. This chapter will review the fundamentals of in vivo confocal imaging and the clinical applications in general and surgical dermatology.

Dermatology↗
PubMed1990

Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy.

A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single-stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly-(A)+ RNA was performed with a poly-biotin-d(U)-tailed oligo-d(T) probe. Bound probe was detected using streptavidin-fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di-amidine-2-phenyl indol. Multi-parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly-(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly-(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.

Animals↗
PubMed1998

[Keratinocyte density of the cornea in vivo. Automated measurement with a modified confocal microscopy MICROPHTHAL].

BACKGROUND: The MICROPHTHAL is a confocal slit light scanning microscope for a non-invasive in-vivo examination of corneal structures of human eyes. With this instrument even thin layers of corneal tissue can be imaged in good quality. Otherwise, blurring of single frames and deviations from the z-axis in video-sequences caused by high speed movements of the eye would normally prevent a measurement the density of keratocytes in the cornea. The goal of the investigation was optical pachymetry, the automatical measurement of the keratocytes density and a 3D-dimensional reconstruction of the central cornea in-vivo under constant imaging conditions. MATERIALS AND METHODS: We developed a low-vacuum suction cup system for stabilizing the eye in front of the microscope objective during the z-scan through the cornea. A stepmotor shifting system for the objective locates inside the suction cup with a central hole was installed underneath them icroscope. Control of this system via computer facilitated shifting the focal plane along the z-axis. The layer images were recorded using a S-VHS-tape and saved on the PC. The digital analysis was performed using a special software to automatically and off-line evaluate the density of keratocytes in combination with the 3D-reconstruction. The software also corrected the background illumination and small axial jitter. After this procedure the keratocytes density and the 3D-reconstruction in 70 images of the z-scan were calculated. We examined 47 corneas of 25 healthy probands. The range of age was 25-56 years. Independent control evaluation of the video sequences were taken manually on an INDIGO HIGH IMPACT workstation. RESULTS: By assign all keratocytes to the corneal measurement volume we found a averaged density of 15,730 cells/mm3 in the central cornea. The averaged thickness of the cornea was 0.556 mm. The control valuation of identical video-sequences on the workstation accomplished the same result of 16,000 keratocytes/mm3, also similar the result of the automatically measurement with the modified software. CONCLUSIONS: This modification of the microscope is a promising in-vivo tool for optical pachymetry and quantitative examination of corneal microstructures. The stabilization effect of the low-vacuum suction cup system in the front of the microscope for computer-controlled valuation of the density profile of keratocytes and the 3D-reconstruction of a central corneal volume element has produced encouraging results. Characterization of pathophysiological changes in the distribution of keratocytes after excimer laser ablation for phototherapeutic or photorefractive keratectomy, for example, can be estimated without pain for the patients.

Adult↗
PubMed2001

Phagocytosis of malarial pigment haemozoin by human monocytes: a confocal microscopy study.

Haem from host erythrocyte (RBC) haemoglobin is polymerized in the digestive organelle of Plasmodium falciparum to haemozoin (HZ), a crystaLline, insoluble substance. Human monocytes avidly ingest HZ that persists undigested for long periods of time, and generates potent bioactive lipid peroxide derivatives. Protein kinase C, an effector of signal transduction, phagolysosome formation and acidification, is inhibited in HZ-fed monocytes. Inability to digest HZ might derive from impairment in phagolysosome formation or acidification. Time-course and extent of HZ phagocytosis and acidification of phagolysosomes were studied by quantitative confocal microscopy. From 180 min until 72 h after the start of phagocytosis approximately 75-79% of the monocytes contained massive amounts of HZ. Coincidence between red (HZ) and green (acidic organelles) fluorescent compartments was very high. Confocal images showed that at 30-60 min after the start of phagocytosis, HZ was preferentially present as separated particles, with full co-localization of red and green fluorescence. Later on HZ-laden phagolysosomes tended to fuse together. In conclusion, phagolysosome formation and acidification were normal in HZ-fed monocytes during the 72-h observation time. The presence of HZ in the phagolysosome, the site of antigen processing, may offer a physical link with immunodepression in malaria.

Animals↗
PubMed1996

Glutamic acid decarboxylase 65 and 67 isoforms in fetal, neonatal and adult porcine islets: predominant beta cell co-localization by light and confocal microscopy.

Glutamic acid decarboxylase (GAD) has been shown to exist as two isoforms with molecular weights of 65 kD (GAD65) and 67 kD (GAD67) in the central nervous system as well as in several non-neuronal tissues, including the pancreatic islets. Recently, this enzyme has been proposed as a key beta-cell autoantigen in insulin-dependent diabetes mellitus (IDDM). In the present study, we used double label light and confocal microscopy to examine the expression of the two GAD isoforms in islet cells of fetal, neonatal and adult porcine pancreas. We also aimed to identify the islet cell-type(s) which co-express GAD. In the adult pig, GAD65 was localized exclusively in most of the beta cells, whereas GAD67, in addition to being present in a majority of the beta cells, was also seen in a proportion of glucagon and somatostatin labelled cells. In the 90-day fetus and the 7-day neonate, while GAD65 was also observed in a majority of beta cells, a proportion of glucagon cells also co-expressed this isoform. The cellular expression of GAD67 in the fetal and neonatal stages was similar to that in the adult. Detailed confocal analysis of GAD65 immunoreactive cells showed a granular cytoplasmic staining, with labelled granules often concentrated in specific perinuclear regions, possibly the Golgi apparatus. In contrast, GAD67 positive cells showed more diffuse cytoplasmic staining. The predominant expression of both the isoforms in porcine beta cells suggests that islet cells from this species may act as a suitable cellular model for study of GAD autoreactivity during the early stages of IDDM.

Animals↗
PubMed1999

Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy.

We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1, 1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C(20)) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3 -phosphoc holine (Bodipy-PC). Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D(T), of the DiI-C(20) was measured by FCS. D(T) values at approximately 25 degrees C ranged from approximately 3 x 10(-8) cm(2)/s in the fluid phase, to approximately 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to approximately 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases. In favorable cases, FCS could distinguish two different values of D(T) in a region of two-phase coexistence on a single vesicle.

Carbocyanines↗
PubMed1991

Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy.

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-ceramide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PK1 porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 micron). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PK1 cells, about 2.5% in 3T3 cells and L5 cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system.

Two methods were compared for determining the concentration of penetrative biomass during growth of Rhizopus oligosporus on an artificial solid substrate consisting of an inert gel and starch as the sole source of carbon and energy. The first method was based on the use of a hand microtome to make sections of approximately 0.2- to 0.4-mm thickness parallel to the substrate surface and the determination of the glucosamine content in each slice. Use of glucosamine measurements to estimate biomass concentrations was shown to be problematic due to the large variations in glucosamine content with mycelial age. The second method was a novel method based on the use of confocal scanning laser microscopy to estimate the fractional volume occupied by the biomass. Although it is not simple to translate fractional volumes into dry weights of hyphae due to the lack of experimentally determined conversion factors, measurement of the fractional volumes in themselves is useful for characterizing fungal penetration into the substrate. Growth of penetrative biomass in the artificial model substrate showed two forms of growth with an indistinct mass in the region close to the substrate surface and a few hyphae penetrating perpendicularly to the surface in regions further away from the substrate surface. The biomass profiles against depth obtained from the confocal microscopy showed two linear regions on log-linear plots, which are possibly related to different oxygen availability at different depths within the substrate. Confocal microscopy has the potential to be a powerful tool in the investigation of fungal growth mechanisms in solid-state fermentation.

Biofilms↗
PubMed2005

Long-term in vivo investigation of mouse cerebral microcirculation by fluorescence confocal microscopy in the area of focal ischemia.

This study was designed to assess that mouse pial and cortical microcirculation can be monitored in the long term directly in the area of focal ischemia, using in vivo fluorescence microscopy. A closed cranial window was placed over the left parieto-occipital cortex of C57BL/6J mice. Local microcirculation was recorded in real time through the window using laser-scanning confocal fluorescence microscopy after intravenous injection of fluorescent erythrocytes and dextran. The basal velocity of erythrocytes through intraparenchymal capillaries was 0.53+/-0.30 mm/sec (n=121 capillaries in 10 mice). Two branches of the middle cerebral artery were topically cauterized through the window. Blood flow evaluated by laser-Doppler flowmetry in two distinct areas indicated the occurrence of an ischemic core (15.2%+/-5.9% of baseline for at least 2 h) and a penumbral zone. Magnetic resonance imaging and histology were used to characterize the ischemic area at 24 h after occlusion. The infarct volume was 7.3+/-3.2 mm(3) (n=6). Microcirculation was repeatedly videorecorded using fluorescence confocal microscopy over the next month. After the decrease following arterial occlusion, capillary erythrocyte velocity was significantly higher than baseline 1 week later, and attained 0.74+/-0.51 mm/sec (n=76 capillaries in six mice, P<0.005) after 1 month, while venous and capillary network remodeling was assessed, with a marked decrease in tortuosity. Immunohistochemistry revealed a zone of necrotic tissue into the infarct epicenter, with activated astrocytes at its border. Such long-term investigations in ischemic cortex brings new insight into the microcirculatory changes induced by focal ischemia and show the feasibility of long-term fluorescence studies in the mouse cortex.

Animals↗
PubMed1999

An immunocytochemical approach to the study of beta-endorphin production in human keratinocytes using confocal microscopy.

Proopiomelanocortin (POMC) is a protein that is posttranslationally processed to yield POMC peptides. The main site of POMC expression is the anterior pituitary lobe but many other sources have been identified. There is evidence that the skin produces POMC peptides, although their roles have not yet been defined. In the skin, regulation of POMC gene expression is known to be hair-cycle dependent, and it is localized to the sebaceous gland. In particular, beta-endorphin, a POMC peptide, has been shown to be modulated by TPA, IL-1 alpha, and ultraviolet radiation in keratinocytes. These results were obtained by examination of POMC mRNA levels using the Northern blot method; beta-endorphin protein production by the Western blot method on cultured cells; and immunocytochemistry for tissue preparations. This report represents an approach to use immunocytochemistry to quantify beta-endorphin production in cultured human keratinocytes. Additionally, we examined whether exposure to 20 mJ ultraviolet B radiation (UVB) and/or UVA could influence beta-endorphin production in these cells. Keratinocytes were grown in monolayers, in serum-free medium, fixed, and incubated with antiserum to whole synthetic beta-endorphin. Fluorescence microscopy was performed with a confocal laser scanning microscope. The integrated level of fluorescence was evaluated in n = 18 +/- 8 individual cells, and this was assumed to be proportional to beta-endorphin content. High variability was observed in the fluorescence intensity among cells. No significant differences between control and UVB- or UVA + UVB-treated cells was found. Similar results were produced by using brefeldin A, a compound that disrupts the secretory pathway, eliminating the possibility that the absence of a difference between beta-endorphin content in the treated and control cells was due to secretion of the peptide into the medium. We conclude that: (1) beta-endorphin or beta-endorphin-like peptides are produced in human keratinocytes and are readily detected by immunocytochemistry; (2) under the conditions tested, UVA and/or UVB did not increase beta-endorphin-like immunoreactivity in these cells.

Cells, Cultured↗
PubMed2001

Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study.

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.

Acridine Orange↗
PubMed2001

Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.

Animals↗
PubMed2006

Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy.

Minute-to-minute control of the release of insulin by pancreatic beta-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events ('transient' versus 'full fusion') are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 beta-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

Animals↗
PubMed2006

High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.

Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.

Algorithms↗
PubMed2002

The application of confocal microscopy to the study of liposome adsorption onto bacterial biofilms.

Confocal laser scanning microscopy has been used to visualise the adsorption of fluorescently labelled liposomes on immobilised biofilms of the bacterium Staphylococcus aureus. The liposomes were prepared with a wide range of compositions with phosphatidylcholines as the predominant lipids using the extrusion technique. They had weight average diameters of 125 +/- 5 nm and were prepared with encapsulated carboxyfluorescein. Cationic liposomes were prepared by incorporating dimethyldioctadecylammonium bromide (DDAB) or 3, beta [N-(N1,N1 dimethylammonium ethane)-carbamoyl] cholesterol (DC-chol) and anionic liposomes were prepared by incorporation of phosphatidylinositol (PI). Pegylated cationic liposomes were prepared by incorporation of DDAB and 1,2-dipalmitoylphosphatidylethanolamine-N-[polyethylene glycol)-2000]. Confocal laser scanned images showed the preferential adsorption of the fluorescent cationic liposomes at the biofilm-bulk phase interface which on quantitation gave fluorescent peaks at the interface when scanned perpendicular (z-direction) to the biofilm surface (x-y plane). The biofilm fluorescence enhancement (BFE) at the interface was examined as a function of liposomal lipid concentration and liposome composition. Studies of the extent of pegylation of the cationic liposomes incorporating DDAB, on adsorption at the biofilm-bulk phase interface were made. The results demonstrated that pegylation inhibited adsorption to the bacterial biofilms as seen by the decline in the peak of fluorescence as the mole% DPPE-PEG-2000 was increased in a range from 0 to 9 mole%. The results indicate that confocal laser scanning microscopy is a useful technique for the study of liposome adsorption to bacterial biofilms and complements the method based on the use of radiolabelled liposomes.

Adsorption↗
PubMed1998

Application of real-time confocal microscopy for observation of living cells in tissue specimens.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) has been a fundamental technique in cell biology. However, most investigations have used cultured or isolated cells as an experimental model, and consequently can provide only limited insight into the mechanisms that operate in tissue in situ. Useful information may be obtained by studying intact tissue specimens. High-speed confocal microscopes that can acquire digital images at video rate have recently been developed. These confocal microscopes which can acquire data in real-time enable [Ca2+]i dynamics of individual cells in intact tissue specimens to be observed. The present paper examines the use of fluorescent microscopy and confocal microscopy for [Ca2+]i imaging of living tissue. We analyzed the dynamics of the duodenal gland, lacrimal gland, intestinal smooth muscles, arterioles, myenteric plexus, and dorsal root ganglion. In these specimens, individual cells exhibited different [Ca2+]i dynamics, and the responses to transmitters/modulators were heterogeneous. In conclusion, real-time imaging provides a useful tool for observing dynamic changes in cells in situ, and it may lead to improve understanding tissue physiology.

Biological Transport↗