Applications of confocal laser scanning microscopy to dental bonding

Groups of photosensitive adult female blackheaded buntings were exposed to various ultrashort days photoperiodic regimes for 60 days in which a fixed 3 h photophase was coupled with dark phases in cycles of 22 to 32 hours duration. One group of buntings was kept in long days (15L:9D) as control. Significant increase in ovarian weight and circulating plasma estradiol concentration was marked in the cycles of 30 h (3L:27D), and 32 h (3L:29D) photoperiodic schedules as well as in control group (15L:9D), whereas there was no response in the cycles of 22 h (3L:19D) and 24 h (3L:21D). It seems that the response to ultrashort day cycles is due to a phase advance or delay in photosensitivity of the response system repeatedly shows coincidence of the external photophase (3 h) with the photoinducible phase of an endogenous circadian rhythm. Therefore, the present result appears to be consistent with Bünning hypothesis suggesting the involvement of an endogenous circadian component in the female blackheaded bunting.     

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.     

Applications of laser scanning confocal microscopy to the observation of human oocytes and embryos

Continuity of care beyond the walls of the acute hospital setting has always been a major emphasis in nursing. There is concern that the care needs of older adults at the time of discharge have been increased by shortened hospital stays. Yet little is known about the specific and changing health care needs of older adults during the early days at home following discharge from acute care, particularly those who are discharged without community referrals. To learn more about the experiences of this population, the College of Nursing at the University of Southern Maine, in collaboration with the Nursing Service Department at Maine Medical Center, conducted a demonstration project. This project involved follow-up home visits to older adults who were discharged to their homes from an acute care setting.     

共焦激光扫描显微镜在冶金中的应用

共焦激光扫描显微镜（CLSM）是普通光学显微镜与激光、计算机相结合的产物,具有比普通光学显微镜更高的分辨率,并且可以实现对样品的分层扫描,进而进行动态原位观察。利用CLSM可以直接观察金属的晶体长大过程,高温相变过程以及凝固过程中第二相粒子的各种行为。本文综述了CLSM系统的组成和原理以及其在冶金领域的应用,如夹杂物的碰撞、聚集、长大,在钢渣界面的扩散过程及在凝固前沿的捕捉/推进行为,析出物的形核析出过程等。     

Detection of human papillomavirus DNA in CaSki and HeLa cells by fluorescent in situ hybridization. Analysis by flow cytometry and confocal laser scanning microscopy

CaSki and HeLa cell lines, isolated from human uterine carcinomas and containing integrated human papillomavirus (HPV) DNA type 16 and 18, respectively were used to evaluate the sensitivity of HPV-DNA detection on suspended cells by fluorescent in situ hybridization using flow cytometry and on corresponding cell deposits using confocal laser scanning microscopy (CLSM). HPV DNAs were detected in cell suspensions with biotinylated DNA probes and revealed with a three-step technique: a rabbit antibiotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. By flow cytometry, HPV DNA was detectable only in CaSki cells which contained about 600 copies of HPV DNA per cell. In HeLa cells, with only 20-50 copies of HPV DNA, flow cytometry could not detect HPV DNA, whereas CLSM permitted visualization of fluorescent labelling of HPV DNA hybrids. Furthermore, CLSM showed good preservation of cellular morphology and the nucleus was clearly recognizable after fluorescent in situ hybridization and counterstaining with propidium iodide. Moreover, this examination confirmed that the fluorescent foci were specifically confined to the cell nuclei.     

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.     

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.     

Cytologic application of p53 overexpression using immunocytochemistry and confocal laser scanning microscopy

OBJECTIVE: p53 Protein plays an important role in cellular growth control. This study investigated p53 protein expression in cell smears of endometrial carcinomas supplemented by confocal laser scanning microscopy (CLSM). STUDY DESIGN: Imprints from surgical specimens of 20 endometrial carcinomas were used. p53 Protein expression was investigated immunocytochemically using the monoclonal antibody pAb1801. Using CLSM, three-dimensional morphology was studied. RESULTS: Of the 20 cases of endometrial carcinoma, 8 stained positively for p53 protein. p53 Showed heterogeneous intranuclear localization, which appeared to be associated with chromatin structure. CONCLUSION: Immunocytochemical detection of p53 overexpression in cell samples is practical, and CLSM has vast potentials in studying the intranuclear arrangement of chromatin.     

The structure of a model pulmonary surfactant as revealed by scanning force microscopy

The structures formed by a pulmonary surfactant model system of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and recombinant surfactant-associated protein C (SP-C) were studied using scanning force microscopy (SFM) on Langmuir-Blodgett films. The films appeared to be phase separated, in agreement with earlier investigations by fluorescence light microscopy. There were smooth polygonal patches of mostly lipid, surrounded by a corrugated rim rich in SP-C. When the films were compressed beyond the equilibrium surface pressure, the protein-rich phase mediated the formation of layered protrusions. The height of these multilamellar structures embodied equidistant steps slightly higher than a DPPC double layer in the gel phase. At the air-water interface too, a high compressibility at low surface tension was indicative of the exclusion of matter. The exclusion process proved to be fully reversible. The present study demonstrates that some of the matter of the model pulmonary surfactant can move in and out of the active monolayer. The SFM images revealed a lipid-protein complex that was responsible for the reversible exclusion of double-layer structures. This mechanism may be important in the natural system too, to keep the surface tension of the alveolar air/water interface constantly low over the range of area encountered upon breathing.     

The sporulation process in Thermomonospora fusca as revealed by scanning and transmission electron microscopy

The sporulation process in the thermophilic actinomycete Thermomonospora fusca was observed by scanning and transmission electron microscopy. As shown by scanning electron microscopy, spores were produced primarily on aerial hyphae and first appeared as bud-like enlargements at the tips of short multibranched sporophores. Young spores were oval to spherical in shape with a smooth surface. As they matured spores enlarged and developed a rough and globular covering, which was quite fragile and easily detached from the spore. This outer layer, as observed by transmission electron microscopy, was thought equivalent to the sheath of other Thermomonospora species. In cross section, mature spores exhibited a thick spore coat underneath the outer globular layer. This spore coat was usually observed as a single layer, but some spores produced a bilayered coat. No multilayered spore coat or spore cortex was observed in the heat-sensitive spores of T. fusca. They were, therefore, shown to be aleuriospores (microcondia), and not endospores.     

低碳钢在高温共焦激光扫描显微镜下马氏体相变的原位观察研究

利用高温共焦激光扫描显微镜,对低碳钢进行了马氏体相变的原位动态观察。结果表明,实验用低碳钢在连续冷却过程中形成板条马氏体,Ms点约为373 ℃,Mf点约为300 ℃。板条马氏体主要在退火孪晶处以及奥氏体晶界及其角隅处形核,或者在先形成的板条处形核,再以60°或120°角向奥氏体晶内生长。板条束的形成也有两种类型,一类以先形成的板条为基准逐步形成彼此平行的板条束,另一类则由先形成的板条触发60°或120°方向的板条。最终构成正三角形、平行四边形等几何形状。     

Tight junctional permeability in living cells: dynamic changes directly visualized by confocal laser microscopy

Confocal microscopy was used to study the tight junctional permeability in living rat parotid and submandibular glands. The interstitial space of the tissue was perfused with medium containing fluorescent tracers Lucifer Yellow (anionic: MW 457), Propidium Iodide (cationic: MW 668) and dextrans labeled with FITC or RITC (anionic and neutral: MW 3K, 10K, 40K, 70 K and 500 K) to monitor whether or not these tracers permeate into the lumen across the junction. In the acini of normal glands, fluorescence was detected in the basolateral space but not in the luminal space up to 30 min. However, when secretion was induced by isoproterenol or carbachol, fluorescence appeared in the luminal space within 2 to 5 min. This did not involve the disruptive changes in tight junction ultrastructure, nor was it irreversible; the luminal fluorescence disappeared again when the secretagogues were removed. Tracers up to MW 40 K for isoproterenol and MW 10 K for carbachol revealed the luminal fluorescence in parotid acini, with little indications of the charge preference characteristics. The luminal fluorescence also appeared by anoxia, enzymatic cell dissociation and the cytochalasin D treatment. It was suggested that the tight junctions in salivary acini dynamically alter their permeability and modulate the passage of large molecules through the paracellular pathway. Oxygen supply, extracellular matrices and cytoskeletons were suggested to influence these regulations.     

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.     

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.     

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.     

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.     

Contribution of confocal laser scanning microscopy to glycochemistry of mouse and rat submandibular glands by single and double lectin staining

The localization of individual glycosidic residues in the mouse and rat submandibular glands was examined using confocal laser scanning microscopy (CLSM). For these organs we tested some procedures of fixation and embedding to better understand the distribution of some lectin-probes inside well preserved secretory cells and observed that fixation and inclusion steps did not influence appreciably the location and intensity of the reactive sites. The fixation mixture of 4% paraformaldehyde, 1% glutaraldehyde and 0.2% picric acid produced the most satisfactory results. In specimens labeled with PNA-, Con A-, LTA-FITC and WGA-, DBA-TRITC lectins, the convoluted granular tubules (CGT) proved to be composed of secretory granules with high-density lectin labeling. The complex organization of secretory glycocomponents within the granule matrix was further resolved by double labeling and dual scanning experiments. Some lectins exhibited colocalization while others displayed differential localization providing information about the occurrence of O- and N-linked glycoconjugates. The CLSM technique applied to fluorochrome-conjugated lectins also revealed a more marked dimorphism in the rat rather than in the mouse submandibular gland. In particular, the male rat submandibular gland was found to consist of CGT heterogeneous cell populations, while the mouse submandibular gland did not show glycochemical differences between cells. Female rats exhibited a lectin profile very different from that of female mouse.