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PubMed1998

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 degrees C gave higher immobilization yield than freezing at -75 or -8 degrees C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy.

Chromatography↗
PubMed1993

The application of confocal microscopy to the study of living systems.

A unique tandem confocal microscope (TSCM) has been developed that permits noninvasive imaging in vivo of the eye and many other organ systems in real time in situ. The application to the study of microphysiological processes in vivo is described and illustrated for the cornea, kidney, liver, epididymis, muscle, and adipose tissue. Novel applications are shown for studying the healing of wounds in four dimensions (x, y, z, t) in single animals over time at the cellular level. Application to clinical diagnostic use in humans is also demonstrated. When combined with Laser Scanning Confocal fluorescence microscopy, the TSCM offers a unique new imaging paradigm for experimental biology and medicine with great potential for use in neuroscience and many other disciplines.

Animals↗
PubMed1991

Demonstration by confocal microscopy that unliganded overexpressed glucocorticoid receptors are distributed in a nonrandom manner throughout all planes of the nucleus.

Mouse glucocorticoid receptors (GR) that are over-expressed in Chinese hamster ovary (CHO) cells behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine the localization of the overexpressed GR within the CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled pattern throughout all planes of the nucleus. The receptor is not present in nucleoli and shows no preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each plane of the nucleus demonstrates clearly that there are regions that do not contain receptor; thus, the distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is no detectable change in receptor distribution. Overexpressed receptors that have either no hormone-binding activity or no DNA-binding activity because of point mutations localize in the same mottled pattern as the wild-type receptor. These observations are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded state and proceed to loci distributed throughout the nucleus, where it is retained in an inactive docking complex until the binding of hormone triggers its progression to high affinity sites where the primary events in transcriptional activation occur. As there is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site where the primary events in transcriptional activation occur.

Animals↗
PubMed1994

Quantification and localization of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) molecules on bronchial epithelial cells of asthmatics using confocal microscopy.

An increased expression of HLA-DR and ICAM-1 molecules on bronchial epithelial cells has been observed in asthmatic patients. The aim of this study was to evaluate the localization and to quantify the spontaneous expression of HLA-DR and ICAM-1 on bronchial epithelial cells recovered by bronchial brushing of nine asthmatics and nine controls. Epithelial cells constituted over 95% of cells recovered as shown using an anti-cytokeratin MoAb. Expression of HLA-DR and ICAM-1 was studied using indirect immunofluorescence and confocal microscopy. The intensity of fluorescence of epithelial cells expressing HLA-DR and ICAM-1 was significantly (P < 0.003) increased in asthmatics. In asthmatics, but not in controls, the expression of both molecules was localized in the cytoplasm on the apicolateral portions of the cells. This study shows an up-regulation in the expression of HLA-DR and ICAM-1 molecules by bronchial epithelial cells from asthmatics and a localization of these molecules within the cell.

Adult↗
PubMed1998

Measurement of enamel remineralization using microradiography and confocal microscopy. A correlational study.

Tooth minerals are lost and regained constantly in a normal, human oral environment. Different methods have been developed to measure this gain and loss in enamel minerals; however, these methods deal with different problems, such as being time consuming or involving the use of X-rays. The aim of this study was to determine if remineralization measured in a thin enamel section (TS) by transversal microradiography (MR) can be reliably monitored by measuring lesion parameters (area, total and average dye fluorescence) on the same TS or on half a tooth (HT) with confocal laser scanning microscopy (CLSM). Thirty-six human enamel specimens were demineralized for 96h, and then half of each specimen was covered with an acid-resistant nail varnish. Specimens were divided into three groups (12/group) and subjected for 20 days to a cyclic remineralization regimen with consisted daily of a 4-hour acid challenge, four 1-min treatment periods with 0, 250 or 1,100 ppm F dentifrice slurries (1:2; dentifrice:water) and 20 h in pooled, human saliva, at room temperature. Specimens were cut and analyzed by MR, then stained with a fluorescent dye (0.1mM rhodamine B) for 1 h and analyzed using CLSM. Both MR and CLSM detected significantly greater remineralization (p<0.05) in the specimens treated with the fluoride-containing dentifrices than in the specimens treated with 0 ppm F. Significant differences were detected between specimens treated witht the fluoride-containing dentifrices by MR and CLSM (HT area and total fluorescence). Statistically significant (p<0.05) Pearson correlation coefficients were calculated between the MR and CLSM data: difference in MR mineral content (DeltaM) versus HT lesion area = 0.71; DeltaM versus HT total fluorescence = 0.70; DeltaM versus HT average fluorescence = 0.61; DeltaM versus TS lesion area = 0.88; DeltaM versus TS total fluorescence = 0.63, and DeltaM versus TS average fluorescence = 0.40. It is concluded that confocal microscopy in either TS or HT may provide valid surrogates (area and total fluorescence) for MR measurements in enamel remineralization studies.

Analysis of Variance↗
PubMed1994

Modulation of Ca2+ release in cultured neonatal rat cardiac myocytes. Insight from subcellular release patterns revealed by confocal microscopy.

It is well established that in heart muscle the influx of Ca2+ through Ca2+ channels during the action potential is the main trigger for Ca2+ release from the sarcoplasmic reticulum (SR), but intact cardiac tissue and single myocytes are also known to exhibit spontaneous Ca2+ release from the SR under a variety of circumstances. Although conditions favoring spontaneous activity have been examined extensively, mechanisms modulating or regulating spontaneous as well as triggered Ca2+ release are still largely unknown. Using the high spatial and temporal resolution of laser-scanning confocal microscopy, we investigated subcellular aspects of spontaneous and triggered Ca2+ release in isolated rat neonatal myocytes loaded with the Ca(2+)-sensitive fluorescent dye fluo 3. Three distinct patterns of spontaneous Ca2+ release were identified: (1) a homogeneous Ca2+ release, presumably corresponding to Ca2+ release during a spontaneous action potential, (2) a focal or spatially restricted Ca2+ release with no or only limited subcellular propagation, and (3) a Ca2+ release propagating as a wave throughout the entire cell. Pharmacologic tools that interfere with the SR revealed that all release types were critically dependent on the Ca2+ release and uptake function of the SR. From our results we conclude that the probability, extent, and pattern of Ca2+ release are modulated on the subcellular level. The observed spectrum of release patterns can be explained by a space- and time-dependent variability in the positive feedback of the Ca(2+)-induced Ca(2+)-release mechanism within an individual myocyte. Presumably, this variability depends on the existence of subcellular functional elements of the SR. The actual degree of positive feedback may be modulated locally by the Ca(2+)-loading state of each SR element.

Action Potentials↗
PubMed1990

Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy.

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.

Actinin↗
PubMed2007

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy.

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Animals↗
PubMed1992

Study of precorneal tear film thickness and structure by interferometry and confocal microscopy.

Past measurements of precorneal tear film thickness in rabbits and humans gave values of 4-7 microns. These have been the accepted values. However, measurements presented in this report are much higher. Earlier techniques may have changed film thickness or not have fully included the layer containing mucus. Little is known about the mucus component. Thickness was measured in freshly killed animals of 10 species using two independent optical techniques. Coherent light was reflected from eyes and thickness was determined from spacing of interference fringes. Clear images of tissue structure were obtained with confocal microscopy, the tear film was visually identified, and thickness measured. Measurements by the two methods were closely correlated in each animal. No film was detected in trout or carp. In other species, thickness ranged from 9 microns in frogs to 15 microns in gerbils. Values are significantly larger than earlier estimates. Film thickness in three living rabbits was not significantly different from that measured shortly after death. There was no variation in thickness at five positions across the cornea in three species. Mucus content of rat tear film was examined by measuring thinning after application of 4, 8, and 20% (weight/volume) acetylcysteine, a mucolytic agent. Thickness was considerably reduced compared to controls, and the film almost completely removed by the highest concentration. The authors propose that the film is largely composed of mucus, not free aqueous solution. Our methods include such layers, and measurements are correspondingly larger than earlier ones.

Animals↗
PubMed1994

An examination of the micromechanics of failure of bone and antler by acoustic emission tests and Laser Scanning Confocal Microscopy.

Bone fails as a result of damage accumulation in the form of microcracks. Antler is in essence less mineralized bone, but is much tougher than ordinary bone. Failure through damage accumulation also happens in antler, so it is clear that antler's microcracks must be pervasive and that natural selection has produced mechanisms for this material which make it more damage-tolerant. We have examined the development of damage in bone and antler by cyclic loading in three-point bending. We have also conducted acoustic emission studies on these two materials during monotonic tensile loading, and used Laser Scanning Confocal Microscopy (LSCM) to observe microcracks in samples which had been mechanically damaged. We found that diffuse microcracking coincides with the appearance of a macroscopic region of 'yield' in the stress/strain curve of bone and antler; it is related to the local stress (or strain) field that microcracks interact with the structure (particularly in antler). Both acoustic emissions studies and LSCM observations indicated that antler may possess additional modes of stable micromechanical failure compared with ordinary bone.

Animals↗
PubMed2004

Laser scanning confocal microscopy for in situ monitoring of alkali-silica reaction.

Alkali-silica reaction (ASR) occurs in concrete between reactive siliceous components in the aggregate and the strongly alkaline pore solution, resulting in the formation of a potentially expansive gel product. Lithium additives have been shown to reduce expansion associated with ASR, but the mechanism(s) by which lithium reduces expansion have not been understood. Therefore, development of an in situ method to observe reactions associated with ASR is highly desirable, as it will allow for non-destructive observation of the reaction product formation and damage evolution over time, as the reaction progresses. A technique to image into mortar through glass aggregate by laser scanning confocal microscopy (LSCM), producing three-dimensional representations of the sample was developed. This LSCM technique was utilized to monitor the progress of alkali-silica reaction in mortar samples prepared with alkali-reactive glass aggregate both in the presence and in the absence of lithium additives: LiNO3, LiCl or LiOH. The method proved to be effective in qualitatively monitoring crack formation and growth and product formation, within cracks and at the paste/aggregate interface. In particular, dendritic products were observed at the paste/aggregate interface only in those samples containing lithium, suggesting that these products may play a role in ASR mitigation.

Journal Article↗
PubMed2003

Quantification of staphylococcal-collagen binding interactions in whole blood by use of a confocal microscopy shear-adhesion assay.

In bloodborne staphylococcal infections, bacteria and platelets often combine, forming thrombi on the subendothelium, where collagen is exposed. In this process, the collagen serves as a potential binding surface for Staphylococcus aureus. However, the extent and importance of S. aureus-collagen binding interactions in the development of infected thrombi is uncertain. We quantified S. aureus adhesion to collagen in a whole-blood suspension under defined physiologically relevant fluid shear conditions. S. aureus-collagen binding interactions, mediated by both the S. aureus collagen adhesin (CNA) and protein A-von Willebrand factor (vWf), were evaluated using mutant strains and antibody-blocking techniques. The position of adherent bacteria (at the collagen surface or above the surface in the platelet aggregate) was measured using confocal laser microscopy. Results demonstrated significant CNA-collagen interactions and protein A-vWf-collagen binding interactions under physiological shear conditions. We conclude that collagen binding interactions are important in the development of infected thrombi.

Adhesins, Bacterial↗
PubMed1990

Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analog in rat hepatocyte doublets.

To characterize the poorly understood mechanisms of intracellular transport of bile acids, fluorescein isothiocyanate-glycocholate was synthesized and its ring-OH-linked structure established by fast atom bombardment, mass spectroscopy and 13C nuclear magnetic resonance. Biliary secretion of fluorescein isothiocyanate-glycocholate and [14C]-labeled glycocholate in rats was similar, in contrast to the biliary secretion of sodium fluorescein and methylamine-conjugated fluorescein isothiocyanate, which are non-bile acid organic anions. After incubation of cultured hepatocyte doublets with fluorescein isothiocyanate-glycocholate, serial quantitative image analysis of fluorescence was performed in the cellular cytoplasm, perinuclear zone and bile canaliculus. Uptake of fluorescein isothiocyanate-glycocholate into the cytoplasm was inhibited by removal of sodium from the medium and by addition of glycocholate or taurocholate. After preincubation with colchicine, but not lumicolchicine, the proportion of perinuclear to cytoplasmic fluorescein isothiocyanate-glycocholate increased during incubation with fluorescein isothiocyanate-glycocholate. Neither fluorescein isothiocyanate-glycocholate uptake nor canalicular secretion was affected. By confocal microscopy the perinuclear zone containing fluorescein isothiocyanate-glycocholate was identified as the Golgi apparatus by fluorescent colocalization with C6-NBD-ceramide, which specifically identifies the Golgi apparatus. We conclude that colchicine inhibits fluorescein isothiocyanate-glycocholate transfer from the Golgi apparatus to the bile canaliculus. These results suggest that intracellular bile acid transport involves microtubule-dependent vesicular movement from the Golgi apparatus to the bile canaliculus. The role of this process in normal physiology is uncertain.

Animals↗
PubMed1995

Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

Antibodies, Monoclonal↗
PubMed2003

Time series analysis of transmesothelial invasion by endometrial stromal and epithelial cells using three-dimensional confocal microscopy.

OBJECTIVE: To evaluate endometrial adhesion and invasion of peritoneal mesothelium. DESIGN: Descriptive study using confocal laser-scanning microscopy. SETTING: University-based laboratory. PATIENT(S): Women undergoing surgery for benign conditions. INTERVENTION(S): Fluorescence-labeled peritoneal mesothelial cells (PMCs) were grown on coverslips. Fluorescence-labeled endometrial stromal cells (ESCs) and epithelial cells (EECs) and myometrial cells (Myos) were plated on the PMCs. Cultures were examined at 1, 6, 12, and 24-27 hours with differential interference contrast and confocal laser-scanning microscopy. MAIN OUTCOME MEASURE(S): Demonstration of adherence and invasion of endometrial cells through peritoneal mesothelium. RESULT(S): At 1 hour, there was adherence of the ESCs, EECs, and Myos on the perimeter of PMCs. There was no invasion by the Myos. By 6 hours, ESCs and EECs spread over the surface of the PMCs and extended cell processes through PMC junctions. Extension of pseudopodia under the PMCs followed. By 12 hours, there was vacuolization and lifting of PMCs that had been undermined by endometrial cells. CONCLUSION(S): This is the first time-phase study to demonstrate adherence and the process of invasion of endometrial cells through the mesothelium. The application of three-dimensional confocal laser-scanning microscopy is a novel technique that can be used to further examine mechanisms involved in the pathogenesis of the early endometriotic lesion.

Adult↗
PubMed2005

Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.

The recent success of pancreatic islet transplantation has generated considerable enthusiasm. To better understand the quality and characteristics of human islets used for transplantation, we performed detailed analysis of islet architecture and composition using confocal laser scanning microscopy. Human islets from six separate isolations provided by three different islet isolation centers were compared with isolated mouse and non-human primate islets. As expected from histological sections of murine pancreas, in isolated murine islets alpha and delta cells resided at the periphery of the beta-cell core. However, human islets were markedly different in that alpha, beta, and delta cells were dispersed throughout the islet. This pattern of cell distribution was present in all human islet preparations and islets of various sizes and was also seen in histological sections of human pancreas. The architecture of isolated non-human primate islets was very similar to that of human islets. Using an image analysis program, we calculated the volume of alpha, beta, and delta cells. In contrast to murine islets, we found that populations of islet cell types varied considerably in human islets. The results indicate that human islets not only are quite heterogeneous in terms of cell composition but also have a substantially different architecture from widely studied murine islets.

Animals↗
PubMed2002

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved.

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 micro m and axial resolution is 6 micro m. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s-1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.

Animals↗
PubMed1997

Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells.

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.

DNA, Viral↗