Studying arsenic trioxide-induced apoptosis of Colo-16 cells with two-photon and confocal laser scanning microscopy

With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and Fluo 3-AM, we simultaneously observed arsenic trioxide (As2O3)-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+)i within human skin squamous carcinoma cells (Colo-16 cells). Our results indicated that As2O3 induced (Ca2+)i elevation and ROS production within Colo-16 cells, and both (Ca2+)i elevation and ROS production were involved in the apoptosis of Colo-16 cells. These results suggested that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multi-parameter observation of As2O3- induced apoptosis at the single cell level.Key words: Two-photon laser scanning microscopy, confocal laser scanning microscopy, human skin squamous carcinoma cells (Colo-16 cells), arsenic trioxide, apoptosi

Assessment of Food Ingredient Functionality using Laser Microscopy

End of Project ReportThe objectives of this project were, to establish a confocal microscopy facility at Moorepark, to develop suitable methodology for the examination of food products and ingredients, to apply confocal microscopy techniques to food research projects and to use the above technological expertise for commercial applications in the Irish Food Industry. The confocal laser scanning microscopy (CLSM) facility is now established and is fully integrated into the Teagasc research program at Moorepark. The new Confocal Microscopy Service has attracted significant commercial interest and client work is expanding. Results show that confocal laser scanning microscopy is a valuable technique for assessing the functionality of food ingredients in a wide range of food products, as well as being a powerful problem-solving tool. Work is ongoing to develop further specific ingredient localisation techniques, and to promote commercial awareness of the service. Confocal laser scanning microscopy offers a unique contribution to product research and development in the Irish food industry.Department of Agriculture, Food and the Marin

Application of Autofluorescence for Confocal Microscopy to Aid in Archaeoparasitological Analyses

Confocal laser scanning microscopy (CLSM) was used to examine archaeoparasitological specimens from coprolites associated with La Cueva de los Muertos Chiquitos (CMC) located near present-day Durango, Mexico. The eggs for 4 different types of parasites recovered from CMC coprolites were imaged using CLSM to assist with identification efforts. While some of the parasite eggs recovered from CMC coprolites were readily identified using standard light microscopy (LM), CLSM provided useful data for more challenging identifications by highlighting subtle morphological features and enhancing visualization of parasite egg anatomy. While other advanced microscopy techniques, such as scanning electron microscopy (SEM), may also detect cryptic identifying characters, CLSM is less destructive to the specimens. Utilizing CLSM allows for subsequent examinations, such as molecular analyses, that cannot be performed following SEM sample preparation and imaging. Furthermore, CLSM detects intrinsic autofluorescence molecules, making improved identification independent of resource and time-intensive protocols. These aspects of CLSM make it an excellent method for assisting in taxonomic identification and for acquiring more detailed images of archaeoparasitological specimens

Histometric data obtained by in vivo confocal laser scanning microscopy in patients with systemic sclerosis

BACKGROUND: It would be a benefit if time-saving, non-invasive methods could give hints for diagnosing systemic sclerosis. To investigate the skin of patients with systemic sclerosis using confocal laser scanning microscopy in vivo and to develop histometric parameters to describe characteristic cutaneous changes of systemic sclerosis observed by this new technique, we conducted an exploratory study. MATERIALS AND METHODS: Fifteen patients with systemic sclerosis treated with extracorporal photopheresis were compared with 15 healthy volunteers and 10 patients with other disorders also treated with extracorporal photopheresis. All subjects were investigated using confocal laser scanning microscopy in vivo. RESULTS: Micromorphologic characteristics of skin of patients with systemic sclerosis and measuring parameters for melanisation, epidermal hypotrophy, and fibrosis for dislocation of capillaries by collagen deposits in the papillary dermis were evaluated. An interesting finding was an increased thickness of the tissue in the dermal papillae superior to the first dermal papilla vessel. It was also possible to reproduce characteristic histologic features by confocal laser scanning microscopy in vivo. Histometric parameters for fibrosis and vascular features developed in this study showed significant differences in patients with systemic sclerosis compared to controls. CONCLUSIONS: Although the predominant histopathological features in systemic sclerosis are findings of the reticular dermis and the subcutis, and in histopathological investigation the epidermis seems to remain unaffected by the disease, we have demonstrate some characteristic differences in the epidermis and papillary dermis by confocal laser scanning microscopy in vivo. Some of them have not been described so far. However, to use this technique as a tool for diagnosis and/or staging of systemic sclerosis, further studies are needed investigating the sensitivity and specificity of the histometric parameters developed in this study

AFM, ESEM, TEM, and CLSM in liposomal characterization: a comparative study

An outstanding aspect of pharmaceutical nanotechnology lies in the characterization of nanocarriers for targeting of drugs and other bioactive agents. The development of microscopic techniques has made the study of the surface and systems architecture more attractive. In the field of pharmaceutical nanosystems, researchers have collected vital information on size, stability, and bilayer organization through the microscopic characterization of liposomes. This paper aims to compare the results obtained by atomic force microscopy, environmental scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy to point out the limits and advantages of these applications in the evaluation of vesicular systems. Besides this comparative aim, our work proposes a simple confocal laser scanning microscopy procedure to rapidly and easily detect the liposomal membrane

Applications of Confocal Laser Scanning Microscopy (CLSM) in Foods

Much of the work in the area of physical properties of fats is aimed at determining the relationship among triglyceride structure, crystal properties, crystallization conditions, and macroscopic properties of fats. In finished product containing fat, some of these many macroscopic properties include spredability of margarine, butter and spreads; snap of chocolate; blooming of chocolate; and graininess, smoothness, mouthfeel, water binding, and emulsion stability of spreads [1]. Plastic fats consist of a crystal network in a continuous oil matrix. Many articles in the past have been focused on establishing relationships between lipid composition or polymorphism and macroscopic properties of fats without much consideration of the microstructure of the fat crystal network. Germane to a thorough understanding of plastic fat rheology is a characterization of its microstructure. Not including microstructure as a variable will lead to failure in the prediction of macroscopic properties. In many other non fat or low fat products macroscopic properties depend on their structural organization. Emulsion stability, which is one of the most important physical properties of multiple-phase systems, is strongly determined by oil droplet size and interactions among components that determine spatial distribution of lipid and aqueous phases. Thus, control of food properties for various applications requires a better understanding of the relationships between the food microstructure and macroscopic properties.Fil: Rincón Cardona, Jaime A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; ArgentinaFil: Huck Iriart, Cristián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Herrera, Maria Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Studies of the microstructure of polymer-modified bitumen emulsions using confocal laser scanning microscopy

Polymer-modified bitumen emulsions present a safer and more environmentally friendly binder for enhancing the properties of roads. Cationic bitumen emulsion binders containing polymer latex were investigated using confocal laser scanning microscopy. The latex was incorporated into the bitumen emulsion by using four different addition methods and all emulsions were processed with a conventional colloid mill. The emulsion binder films were studied after evaporation of the emulsion aqueous phase. We show how the microstructure and distribution of the polymer varies within the bitumen binder depending on latex addition method, and that the microstructure of the binder remains intact when exposed to elevated temperature. It was found that a distinctly fine dispersion of polymer results when the polymer is blended into the bitumen before the emulsifying process (a monophase emulsion). In contrast, bi-phase emulsion binders produced by either post-adding the latex to the bitumen emulsion, or by adding the latex into the emulsifier solution phase before processing, or by comilling the latex with the bitumen, water and emulsifier all resulted in a network formation of bitumen particles surrounded by a continuous polymer film. The use of emulsified binders appears to result in a more evenly distributed polymer network compared to the use of hot polymer-modified binders, and they therefore have greater potential for consistent binder cohesion strength, stone retention and therefore improved pavement performance

Metal free graphene oxide (GO) nanosheets and pristine-single wall carbon nanotubes (p-SWCNTs) biocompatibility investigation: a comparative study in different human cell lines

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests