A five-dimensional imaging technique to resolve objects in three-dimensional space, spectrum and time has been developed using a single diffractive optical element consisting of a quasi-random array of pinholes. The object space and spectrum were sampled by axially scanning a point object with different wavelengths along the optical axis and recording the corresponding spatio-spectral signatures. The recorded spatio-spectral signatures were catalogued and stored in a library. A thick object emitting light at different wavelengths was placed in the object space within the axial boundaries of the library and illuminated by light sources within the spectral boundaries of the spatio-spectral library and a single object intensity pattern was recorded. The spatio-spectral images of the object were reconstructed by processing the object intensity pattern and the spatio-spectral library in the computer. Depth-wavelength reciprocity was exploited to see colour from depth and depth from colour. A computational approach utilizing the scaling factors in image formation was used to synthesize the spatio-spectral signatures. The application of depth-wavelength reciprocity and the synthesis of spatio-spectral signatures accelerated the training procedure leading to a faster multidimensional multispectral imaging technology in comparison to the existing coded aperture holography techniques. The developed technology was implemented for spatio-spectral-temporal imaging of a fast transient event such as a spark with a temporal resolution of 40 μs. A single camera shot of the spark recorded by a monochrome high-speed camera was converted into intensity cube data along depth for different wavelengths.
Fresnel incoherent correlation holography (FINCH) is a super-resolution incoherent imaging technique built on the principle of self-interference. In FINCH, the object wave is split into two, modulated by diffractive lenses with different focal distances and interfered to generate the hologram. At least three holograms with different phase-shifts between the two object waves were recorded and combined to synthesize a complex hologram. The different planes of the object were reconstructed by numerically propagating the complex hologram to one of the focal planes of the diffractive lenses. FINCH, in general, has a higher lateral resolution but lower axial and temporal resolutions than direct imaging. A new approach inspired by guide-star holography techniques is applied to FINCH. In this study, two diffractive lens functions with different focal lengths have been spatially multiplexed into one using a binary quasi-random function and fabricated using electron beam lithography. In the first step, the point spread hologram library was recorded by axially scanning a point object along the optical axis. In the next step, an object hologram was recorded. The three-dimensional image of the object was reconstructed using a cross-correlation between the point spread hologram library and the object hologram. The modified approach increased the time resolution of FINCH by three times, improved the axial resolving power and enabled the development of the most compact version of FINCH. We believe that FINCH can be applied for monitoring bacterial cell shape during contact with mechanically biocidal nanotextured surface and light matter interactions.
Infrared (IR) microspectroscopy is a powerful molecular fingerprinting tool widely used for the identification of structural and functional composition of biological and chemical samples. The IR microspectroscopy beamline at the Australian Synchrotron can be operated either with a single-point narrow-band mercury cadmium telluride (MCT) detector or a focal plane array (FPA) imaging detector with 64 × 64 pixels. For the implementation of indirect nonscanning imaging technology, the system was operated with the FPA detector. In this study, we propose an indirect IR imaging technique based on the principles of correlation optics using diffractive optical elements such as random pinhole array (RPA) and Fresnel zone plate (FZP). The spatial and spectral variations of point spread functions (PSFs) of the RPA and FZP were simulated for the synchrotron configuration. Intensity responses for 2D objects were simulated using the same simulation conditions and reconstructed using Lucy-Richardson algorithm. Fabrication of diffractive elements for IR wavelengths is often a challenging task as the IR transparent material substrates, such as barium fluoride and calcium fluoride, are highly susceptible to thermal shocks and brittle by nature. The diffractive elements were fabricated by ablating directly on a 100 nm thick gold coated substrate using femtosecond laser pulses. The simulation results and the fabrication outcomes demonstrate the feasibility of indirect imaging at the synchrotron IR beamline.
The presence of an infection in a wound site is typically diagnosed based on the external appearance, such as redness, swelling, odour, and/or loss of function. However, this can lead to inaccurate and untimely diagnoses, since an infection might be present without obvious symptoms. This would commonly require removal of any dressing that might be present, which can cause further pain to the patient. Therefore, there is a need for more precise methods of detecting infections, with minimal effects to the patient. Comparison of temperature differences between infected tissue and healthy tissue shows an increase ranging from 3-4 °C, while normal skin has a temperature gradient of ±1 °C. Hence, monitoring temperature of wounds can be used to detect the presence of an infection. Nanodiamonds (NDs) containing negatively charged nitrogen-vacancy (NV-) centres are capable of monitoring changes in temperature with minimal influence by environmental factors such as pH, ion concentration or molecular interaction. This study looks at encapsulating these NDs into silk fibres for use as a wound dressing that can monitor temperature changes in the wound, without requiring the removal of the dressing. To further enhance the wound healing and anti-bacterial properties, curcumin was also incorporated into the silk fibres. Curcumin is one of the active ingredients in turmeric and is known to significantly enhance wound healing through its anti-inflammatory and antibacterial properties. This study used this curcumin-nanodiamond-silk hybrid wound dressing to investigate the healing capabilities and temperature sensing properties for use as a wound dressing.
This work reports nanodiamond-silk membranes as an optical platform for biosensing and cell growth applications. The hybrid structure was fabricated through electrospinning and mimics a 2D scaffold with high porosity. The negatively charged nitrogen vacancy (NV-) centres in diamond exhibits optically detected magnetic resonance (ODMR), which enables sensing of temperature variations. The NV- centre, as reported in literature, provides a shift of 74 kHz in the ODMR frequency per degree rise in temperature. For our hybrid membranes, we have however observed that the embedded NV- centre provide a greater shift of 95±5 kHz/K in the ODMR frequency. This higher shift in the frequency will result in improved temperature sensitivity enabling the tracking of thermal variations in the biologically relevant window of 25-50 ºC. The thermal conductivity of silk and diamond-silk hybrid will be explored to investigate this enhanced temperature sensing ability of diamond. The hybrid diamond-silk membranes are found to be hydrophilic with a contact angle of (65±2)º. The biocompatibility of the membranes is tested both in vitro in skin keratinocyte (HaCaT) cells and in vivo in a live mouse wound model. The membranes did not induce any toxicity to the cell growth and survival. Moreover, we observed resistance towards the growth and attachment of bacteria.
The adsorption of five proteins with very different molecular characteristics, i.e. α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin, has been characterized using quantitative fluorescence measurements and atomic force microscopy. It has been found that the 'combinatorial' nature of the micro/nano-channels surface allows for the increased adsorption of molecularly different proteins, comparing with the adsorption on flat surfaces. This amplification increases for proteins with lower molecular surface that can capitalize better on the newly created surface and nano-environments. Importantly, the adsorption on micro/nano-fabricated structures appears to be less dependent on the local molecular descriptors, i.e. hydrophobicity and charges, due to the combinatorialization of the nano-areas presented to the proteins. The amplification of adsorption is important, ranging from 3- to 10-fold, with a higher amplification for smaller, globular proteins.
Investigation of protein-polymeric surface interaction requires reliable practical techniques for evaluation of the efficiency of protein immobilization. In this study the efficiency of protein immobilization was evaluated using three different techniques: (1) protein-binding assay with fluorescent detection and (2) quantification, and (3) atomic force microscopy. This approach enables us to rapidly analyse the adsorption properties of different proteins. The comparative physico-chemical adsorption of α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin in the micro-wells fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm) deposited on a Poly(methyl ethacrylate) film has been studied. Correlations were observed between the quantitative and qualitative differences depending on both protein and polymeric surface hydrophobicity.
We recently described a technique to fabricate shallow (< 50 nm) microstructures on PMMA surface for use in multianalyte protein micro-assay based on the ablation of a top thin gold layer using pulsed nitrogen laser (337 nm). In the present study, AFM has been used to investigate the surface characteristics and to provide physical insights into the formation of these complex microstructures. It has been shown that lateral diffusion of the heat generated during the gold ablation extended to ca. 3 μm on either side of the laser focal spot (ca. 5μmm wide), effectively ablated the gold layer and created shallow regions of ca. 20 nm. The heat also created a depression (ca. 5 μm wide) in the polymer region at the laser spot, and a hump, that increased in height with laser dose, at the center of the depression. It is suggested that volume shrinkage caused by stress relaxation and material redistribution, and volume expansion caused by fragmentation of the polymer are responsible mechanisms. Chemical changes also occurred resulting in the middle zone of the microstructure, which corresponds to the central hump, being hydrophobic, whereas the outer zone was hydrophilic. It is suggested that degraded hydrophilic products may be present in the outer zone, whereas the middle zone may contain smaller hydrophobic fragments due to more advanced fragmentation. The variation in the morphology and surface chemistry in the shallow microstructures effectively 'combinatorialize' the surface properties of the microstructures, thus facilitating the patterning of different proteins.
Extracellular polymeric substances (EPS) secreted by bacteria have a key role in adhesion and aggregation of bacterial cells on solid surfaces. In the present study, atomic force microscopy (AFM) has been used to study the adhesion propensity of bacterial strain St. guttiformis, and the ultrastructure and distribution of the EPS materials, on hydrophobic poly(tert-butylmethacrylate)(PtBMA) and hydrophilic polystyrene maleic acid (PSMA) surfaces. The results showed that bacterial attachment to the PSMA surface over incubation periods of 24-72 h was insignificant, whereas there was a strong propensity for the bacterial cells to attach to the PtBMA surface, forming multi-layered biofilms. For the PSMA surface, planktonic EPS adsorbed onto the polymeric surface and formed a continuous surface layer. For the PtBMA surface, non-contact mode imaging revealed that capsular EPS on the cell surface exhibited granular structures with the lateral dimensions of 30-50 nm and the vertical roughness of 7-10 nm. Lateral force imaging showed inter-connected elongated features which had lower frictional property compared to the surrounding EPS matrix, suggesting possible segregation of hydrophobic fractions of the EPS materials. The planktonic EPS adsorbed onto the PtBMA surface also showed similar nanometer-scale granular structures and could form stacks up to 150 nm in height. However, lateral force imaging did not show frictional differences, as in the case of capsular EPS. This is attributed to possible differences in the composition of the two EPS materials, and/or greater deformation of the planktonic EPS in the contact imaging mode which may obscure the fine surface features.
The immobilization efficiency of the complexes of oligonucleotide/poly(L-lysine) onto Poly(Styrene/Maleic Acid), PSMA, and Poly(Styrene/Maleic Anhydride), PSMAA, has been investigated using X-ray photoelectron spectroscopy and atomic force microscopy (AFM) in conjugation with fluorescence-based measurements of DNA attachment. A mono-molecularly thin layer of either electrostatically or covalently (via amide bond) coupled poly(L-lysine) (PL) allows the “switching” of the chemistry from a COOH-based to NH2-based one. The COOH-based chemistry has the advantage of a high yield of reaction but the disadvantage of a low surface concentration of DNA molecules (negative-negative electrostatic exclusion) whereas the NH2-based chemistry provides a higher surface concentration (positive-negative electrostatic attraction) but has a lower yield of covalent binding reaction. The immobilization efficiency of covalently coupled 26-mer oligonucleotides/poly(L-lysine) to polymeric surfaces was estimated as 0.3-0.5 x1012 molecules/mm2 for both polymeric surfaces studied. The electrostatic adsorption of poly(L-lysine)/oligonucleotides onto PSMA and functionalized PSMAA surfaces yielded 0.5 x 1011 and 0.1 x 1010 molecules/mm2, respectively. Although this mode of attachment is not “covalent binding” per se, the evidence is provided that this attachment is strong enough to withstand PCR cycles. The properties of these oligonucleotide/poly(L-lysine) complexes make them promising candidates for DNA-DNA hybridisation assays and PCR.
The immobilization and hybridization of amino-terminated oligonucleotide strands to cyclo-olefin-copolymer (COC) and polycarbonate (PC) surfaces have been investigated for potential application in micro-PCR devices. The oligonucleotides were covalently bound to the plasma-treated COC and PC surfaces via an N-hydroxy-sulfosuccinimide (NHSS) intermediate. Analysis by AFM showed that the oligonucleotides were present on the surfaces as lumps, and that the size, both vertically and laterally, of these lumps on the COC surface was larger compared to the PC surface. The immobilization efficiency of the former was also higher (15.8 x 1012 molecules / cm2) compared to the latter (3.3 x 1012 molecules / cm2). The higher efficiency of the COC surface is attributed to the more effective NHSS-functionalization and its higher surface roughness. Subsequent hybridization doubled the height of the lumps, while the lateral dimensions remained essentially unchanged. This is explained in terms of organization of the long probe strands used on the surface as flexible, coil-like polymer chains, which allow the complementary oligonucleotides to bind and increase the height of the lumps. The AFM frictional images showed that the hybridization had the effect of reversing hydrophilicity of the oligonucleotide lumps from being more hydrophilic to more hydrophobic, consistent with the hydrophilic bases of the probe strands being shielded as a result of hybridization.
Diazonaphthoquinone/novolak (DNQ) photoresist have the property of changing physical-chemical properties during exposure to UV light, which reflects in a change of the polymer hydrophobicity. A combinatorial surface having different exposed area was fabricated, in order to study the influence of hydrophobicity over protein adsorption and EDC-mediated covalent attachment. The results indicate two different behaviours, reflecting a substantial different mechanism of interaction. While protein adsorption decreased following the hydrophobicity decrease, covalent attachment increased, thus reflecting the effectiveness of the covalent mediator, which cross-links the protein to the carboxylic groups that form during exposure. Based on the results of the present work, a combinatorial microarray will be fabricated, to be used in the biosensor field.
Our earlier papers showed that laser gain is possible on the innershell 2s-2p transitions of Ne-like ions. This result was confirmed by more comprehensive calculations. In more recent works it was shown that laser gain is also possible on transitions between highly excited states. We summarize these results and present atomic-kinetic calculations for gain values along the Ne-like sequence for argon, iron, krypton, silver and xenon. For these ions the optimum plasma conditions are found for all possible laser transitions. We have also found some unknown laser transition in Ni-like tantalum and the optimum plasma parameters for laser action on these transitions.
Within accurate atomic kinetic model it is shown that the inversion state of heavy Ne-like ions in plasma is possible at electron densities ne greater than 1023 cm-3 if electron temperature is high enough (Te greater than Eioniz/2). Near optimum plasma conditions are found for Ne-like silver lasers. New effective lasing transitions in Ne- like silver are found. Large ASE effect (gL greater than 100) is predicted for Ne-like silver plasma produced by a powerful short-pulse drive laser.
Energy level populations and spectral line intensities in plasmas are studied using an advanced uniform approach for the calculation of ion energy levels and elementary processes rate coefficients in plasmas. The spectrum of Ne-like argon for the uniform steady-state plasma is calculated for a wide variation of electron temperature and density. Hence the most efficient lasing at 5 3p - 3s transitions with wavelengths 697.6, 860.6, 727.5, 725.7, 670.7 angstroms should be expected at 1019 < ne < 1020 cm-3 and Te approximately 60 eV. It is shown that at electron densities of 1015 < ne < 1021 cm-3 ratios of the strongest 3p - 3s line intensities are sensitive to electron density; their dependence on electron temperature is much less pronounced.
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