Mansour Youseffi

Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Development of a novel liquid crystal based cell traction force transducer system.

Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hookes equation was used to quantify the CTFs by associating Youngs modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Youngs modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1μm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.

Sintering and mechanical properties of prealloyed 6061 Al powder with and without common lubricants and sintering aids

Abstract Physical and mechanical properties for prealloyed 6061 Al powder processed with and without additions of solid and/or liquid lubricants and sintering aids (Pb, Sn, Ag) are presented. For comparison, both vacuum and nitrogen sintering were carried out on as received (gas atomised) and degassed powder compacts pressed at 340 and 510 MPa. Vacuum degassing of the prealloyed powder provided better compressibility and thus higher green densities than those for the as received powder. Highest sintered densities of ∼98–99% of theoretical were obtained for the prealloyed (and degassed) Al compacts by sintering under pure nitrogen with an addition of 0·6 wt-% paraffin wax as solid lubricant or 1·33 vol.-% liquid paraffin, or with a 0·12 wt-%Pb addition as sintering aid and no lubricant. It was found that additions of solid lubricants such as lithium stearate and acrawax to both the premixed (elemental) and prealloyed powders provided reasonable green densities of ∼94·5–95·5% TD, but had deleterious effect on sintered densities and microstructures, particularly under vacuum sintering. Other lubricants such as zinc stearate, stearic acid and liquid paraffin provided similar green densities, but higher sintered densities and less porous microstructures, particularly by sintering under pure nitrogen. The prealloyed compacts sintered under pure nitrogen consistently provided much higher sintered densities than elementally premixed compacts sintered under pure nitrogen or vacuum. It is therefore concluded that both lubricant type and sintering atmosphere will have a major effect on the sintered properties of the 6061 Al powder. Sintering under pure nitrogen resulted in higher sintered densities as compared with vacuum sintering for this grade of Al alloy. Tensile properties of the degassed and vacuum sintered (and T6 tempered) prealloyed powder compacts were higher than those of the equivalent alloy prepared by elemental mixing and comparable with those of the commercial (wrought) 6061 Al alloys.

Supersolidus liquid phase sintering of high speed steels: Part 3: computer aided design of sinterable alloys

AbstractCalculated multicomponent phase diagrams were used to identify high speed steel (HSS) type alloys having the potential to exhibit enhanced sinter ability. The requirement was for an extensive austenite + carbide + liquid phase field. Of the six tungsten and molybdenum based systems studied, Fe–14Mo–C + 4Cr–8Co systems were potentially the most promising. Appropriate compositions were water atomised and additional alloys prepared by blending annealed powders with graphite powders. Powders were compacted to green densities of about 70% theoretical and then vacuum sintered. Sinterability was assessed in terms of sintered densities and microstructures. Alloys containing Fe–13Mo–1·3C, Fe–14Mo–4Cr–1·3C, and Fe–14Mo– 8Co–4Cr–1·4C were sintered to full density at temperatures as low as 1170°C, 70–150 K lower than for existing HSSs. Sintering windows were 20– 30 K, a significant improvement on existing HSSs. As sintered microstructures consisted of angular M6 C carbides dispersed in martensitic matrixes, w...

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Sintering, microstructure, and mechanical properties of PM manganese–molybdenum steels

Abstract The effects of 0·5 wt-%Mo addition on the processing, microstructure, and strength of PM Fe–3·5Mn–0·7C steel are described. Water atomised and sponge irons, Astaloy 1·5Mo, milled ferromanganese, and graphite were the starting powders. During sintering in 75H2 /25N2 or pure hydrogen the dewpoint was controlled and monitored; in particular the effects of improving it from -35 to -60°C were investigated. Faster heating rates (≥20 K min-1), sufficient gas flowrates, milling the ferro alloy under nitrogen, a low dewpoint (<-60°C), and a getter powder can all contribute to the reduction or prevention of oxidation of the manganese, in particular formation of oxide networks in the sintered steels. For 600 MPa compaction pressure densities up to 7·1 g cm-3 were obtained; these were not significantly affected by sintering at temperatures up to 1180°C. The sintered microstructures were sensitively dependent on the cooling rate. Irrespective of the presence of Mo, slow furnace cooling at ∼4 K min-1 resulted in mainly pearlitic structures with some ferrite and coarse bainite, whereas fast cooling at ∼40 K min-1 produced martensite and some retained austenite, very fine pearlite, bainite, and some ferrite. Youngs modulus, determined by tensile and ultrasonic tests, was in the range 110–155 GPa. Sintering with -60°C dewpoint resulted in tensile and transverse rupture strengths of420 and 860 MPa for the Mn steel, rising to 530 and1130 MPa as a result of the Mo addition. This contrasts with strength decreases observed when processing included use of high oxygen containing ferromanganese and sintering with -35°C dewpoint.

Selective laser melting of prealloyed high alloy steel powder beds

This paper presents the results of a recent comprehensive investigation of selective laser melting (slm) of prealloyed gas and water atomised M2 and H13 tool steel powders. The objective of the study was to establish the parameters that control the densification of single and multiple layers with the aim of producing high density parts without the need for infiltration. Powders were processed using continuous wave (CW) CO2 and Nd:YAG lasers. Relationships between alloy composition, powder particle size and shape, flowability, microstructure (phases present, their size, morphology and distribution), track morphology, post scanned density, surface finish and scan conditions (Laser power, spot size and scan speed) are discussed for single track, single layer and multi-layer (up to 25 layers) constructions. Processing with a Nd:YAG laser with powders placed on substrates rather than on a loose powder bed gave more stable builds than with the CO2 laser. Using the Nd:YAG laser densities up to ~90% relative were possible with H13 powder compared with a maximum of ~70% for M2 in multi-layer builds. Maximum density achieved with CW CO2 processing was only ~60%, irrespective of powder composition. The paper compares the processibility of these materials with stainless steel powders processed to higher densities (up to 99% relative) under similar conditions. The results of the work show that a crucial factor for high density processing is melt pool wettability and this is controlled largely by carbon content; low carbon contents producing better wettability, flatter tracks and higher densities. The significance of this observation for the processing high alloy steels by slm will be discussed.

Imaging via widefield surface plasmon resonance microscope for studying bone cell interactions with micropatterned ECM proteins

The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5‐, 10‐, 25‐, 50‐ and 100‐μm‐wide repeat grating. MG63 bone cells were seeded at 50 000 cells per 25 cm2 and cell alignment was determined from micrographs taken at time‐points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin‐patterned cover slips especially to the 50‐ and 100‐μm‐wide patterns, although in this case cells did not position themselves in the middle of each fibronectin‐coated region, but instead aligned to the small features associated with the edges of the fibronectin‐coated regions. Patterned and un‐patterned cells also had quite different morphologies. The un‐patterned cells had a more rounded morphology and lengths of 25 to 35 μm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50–70 μm. The widefield surface plasmon resonance imaging indicated that cells on un‐patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin‐patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin‐coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.

PM processing of elemental and prealloyed 6061 aluminium alloy with and without common lubricants and sintering aids

Abstract A comparison has been made between compaction, sintering, microstructural and mechanical properties of the 6061 aluminium alloy prepared via premixed elemental (EL) and prealloyed (PA) powders (as received and degassed) with and without additions of sintering aids and various solid and/or liquid lubricants. Both EL and PA powders were cold pressed at different pressures, ranging from 250 to 770 MPa, and sintered under vacuum in the range 580–640°C for 30–120 min. and then under pure nitrogen atmosphere for comparison. Vacuum degassing of the PA powder provided better compressibility and thus higher green densities than those for the as received PA or the premixed EL powder compacts pressed at compaction pressures ≥340 MPa. Near full sintered densities of , ∼98%TD were obtained for both EL and PA 6061 Al alloys. Degassed PA Al with 0·6 wt-% paraffin wax (PW) or with only 0·12 wt-%Pb addition as sintering aid and no lubricant, and premixed EL with only 0·12 wt-%Pb addition and no lubricant gave the best optimum properties. It became apparent that additions of some solid lubricants such as lithium stearate (LS) and acrawax to both the premixed EL and PA powders provided reasonable green densities, but had deleterious effect on sintered densities and microstructures, particularly under vacuum sintering. Heating data curves during the sintering cycle, revealed formation of both transient and persistent liquid phases for the EL and mainly supersolidus liquid phase sintering (SLPS) mechanism for the PA. Tensile properties of the degassed, vacuum or nitrogen sintered PA Al alloy in T6 condition were higher than those of the equivalent alloy prepared by EL mixing with the former giving a tensile strength of 330 MPa and 6–8% elongation to failure, which are similar to those of the commercial (wrought) 6061 Al alloys.

Effectiveness of Calcium Nitrite in Retarding Corrosion of Steel in Concrete

Corrosion of steel bars embedded in concrete admixed with 0%, 2% and 4% calcium nitrite (CN), having compressive strengths of 20 and 46 MPa was investigated. Reinforced concrete specimens were immersed in 3% NaCl solutions for 1, 7 and 15 days where 0.4A external current was applied to accelerate the chemical reactions. Corrosion rate was measured by retrieving electrochemical data via potentiodynamic polarization technique. Pull-out tests of reinforced concrete specimens were then conducted to assess the corroded steel-concrete bond characteristics. Experimental results showed that corrosion rate of steel bars and steel-concrete bond strength were dependent on concrete strength, amount of CN added and accelerated corrosion period. As concrete strength increased from 20 to 46 MPa, corrosion rate of embedded steel decreased. The addition of 2% CN to concrete of 20 MPa was not effective in retarding corrosion of steel at long time of exposure. However, the combination of higher strength concrete and 2% or 4% CN appear to be a desirable approach to reduce the effect of chloride-induced corrosion of steel reinforcement. After 1 day of corrosion acceleration, specimens without CN showed higher bond strength in both concrete mixes than those with CN. After 7 and 15 days of exposure, the higher concentration of CN, the higher bond strength in both concrete mixes achieved, except for the concrete specimen of 20 MPa compressive strength with 2% CN that recorded the highest deterioration in bond strength at 15 days of exposure.