Moulded photoplastic probes for near-field optical applications

The inexpensive fabrication of high-quality probes for nearfield optical applications is still unsolved although several methods for integrated fabrication have been proposed in the past. A further drawback is the intensity loss of the transmitted light in the 'cut-off' region near the aperture in tapered optical fibres typically used as near-field probes. As a remedy for these limitations we suggest here a new waferscale semibatch microfabrication process for transparent photoplastic probes. The process starts with the fabrication of a pyramidal mould in silicon by using the anisotropic etchant potassium hydroxide. This results in an inverted pyramid limited by silicon crystal planes having an angle of ~54°. The surface including the mould is covered by a ,1.5 nm thick organic monolayer of dodecyltrichlorosilane (DTS) and a 100-nm thick evaporated aluminium film. Two layers of photoplastic material are then spin-coated (thereby conformal filling the mould) and structured by lithography to form a cup for the optical fibre microassembly. The photoplastic probes are finally lifted off mechanically from the mould with the aluminium coating. Focused ion beam milling has been used to subsequently form apertures with diameters in the order of 80 nm. The advantage of our method is that the light to the aperture area can be directly coupled into the probe by using existing fibre-based NSOM set-ups, without the need for far-field alignment, which is typically necessary for cantilevered probes. We have evidence that the aluminium layer is considerably smoother compared to the 'grainy' layers typically evaporated on free-standing probes. The optical throughput efficiency was measured to be about 10^-4. This new NSOM probe was directly bonded to a tuning fork sensor for the shear force control and the topography of a polymer sample was successfully obtained

Evaluation of DNA damage in murine fibroblasts treated with cigarette smoke condensate

CSC is a complex chemical mixture containing about 4800 compounds, many of them have cytotoxic and mutagenic activities on mammalian cells. Most of these compounds are able to interact with DNA at different levels. Cells may respond to DNA damage by following different pathways, such as the DNA repair processes and the cell cycle and DNA damage checkpoint activation. To the aim to evaluate the biological effects of CSC on cells, alkaline comet assay and flow cytofluorimetry were used to examine DNA damage/repair and cell cycle progression. All experiments were performed by using CSC from standard cigarettes in the range of doses 30-180g/ml and Swiss 3T3 murine fibroblasts. Results obtained by comet assay showed that CSC induces DNA strand breaks, significantly higher after 90 min of treatment than 3hrs. This difference, particularly evident at 100 and 150g/ml, it is probably due to a fast repair that can be explained by the oxidative component of the DNA damage CSC-induced. To clarify these results, further investigations on the evaluation of the oxidative damage are in progress by applying two different methods. one is the detection of the oxidised bases on the DNA by using the modified protocol of Comet assay with FPG and endo III, the second is the analysis of the intracellular levels of ROS, measured as the ability of treated cells to oxidise a fluorogenic dye, is going to be carried out. Previous results of long-term survival showed that cells lose their ability to form colonies in dose-dependent manner, after 24hrs of CSC treatment and 168 hrs of culture. However, the cytofluorimetric analysis showed that a fraction of cells, blocked in G2/M immediately after 24hrs of treatment, are gradually granted to continue the cell cycle, after incubation for further 6hrs in medium CSC-free. Further investigations on the cell cycle alteration are on going

Development of hybrid materials based on hydroxyethylmethacrylate as supports for improving cell adhesion and proliferation

A novel hydrogel based on 2-hydroxyethylmethacrylate and fumed silica nanoparticles is presented. The filler was mixed at increasing amount (3-40% w/w) to the organic monomer, before accomplish thermal polymerization. The hybrid composite materials obtained were characterized as far as concern the physical-chemical stability and sorption behaviour in water and water solutions. The novel hybrid hydrogels were compared to poly(hydroxyethylmethacrylate) (pHEMA) on cytocompatibility and ability to elicit cell adhesion and proliferation. These in vitro assays showed that the first ones were supporting cell growth better then pHEMA, moreover experiments on murine fibroblasts showed improved adhesion and proliferation with the increase of the nanomeric filler content. For a more physiological response, the in vitro tests should match biomaterials with cell populations typical of the implant site. Therefore, in view of future applications of these composites as scaffolds for bone engineering, in a successive step of our research we selected primary cultures of human osteoblasts (OB) as the most appropriate models to study the in vitro performance of these materials. The preliminary results obtained confirmed the remarkable improvement of OB adhesion properties of the new hybrids with respect to pure pHEMA