Søren Hassing

Discriminating isogenic cancer cells and identifying altered unsaturated fatty acid content as associated with metastasis status, using k-means clustering and partial least squares-discriminant analysis of Raman maps.

Raman spectroscopy is a label-free, real-time diagnostic tool that shows great promise in identifying cell differences. We have evaluated the discriminatory power of Raman spectroscopy using a unique model system consisting of two isogenic cancer cell lines derived from the MDA-MB-435 cell line. The two cell lines are equally tumorigenic in mice, but while M-4A4 gives rise to metastasis, NM-2C5 only disseminates single cells that remain dormant in distant organs. Previous comparative proteomic and transcriptomic analyses of the two cell lines have shown that they differ only in the expression level of a few proteins and genes. Raman maps were recorded of single cells after fixation and drying using 785 nm laser excitation. K-means clustering reduced the amount of data from each cell and improved the signal-to-noise ratio of cluster-averaged spectra. Spectra representing the nucleus were discarded as they showed much smaller differences between the two cell lines compared to cytoplasm spectra. Partial least squares-discriminant analysis (PLS-DA) was applied to distinguish the two cell lines. A cross-validated PLS-DA resulted in 92% correctly classified samples. Spectral differences were assigned to a higher unsaturated fatty acid content in the metastatic vs nonmetastatic cell line. Our study demonstrates the unique ability of Raman spectroscopy to distinguish minute differences at the subcellular level and yield new biological information. Our study is the first to demonstrate the association between polyunsaturated fatty acid content and metastatic ability in this unique cell model system and is in agreement with previous studies on this topic.

Tuning the optoelectronic properties of amorphous MoOx films by reactive sputtering

In this letter, we report on the effect of oxygen partial pressure and sputtering power on amorphous DC-sputtered MoOx films. We observe abrupt changes in the optoelectronic properties of the reported films by increasing the oxygen partial pressure from 1.00 × 10−3 mbar to 1.37 × 10−3 mbar during the sputtering process. A strong impact on the electrical conductivity, varying from 1.6 × 10−5 S/cm to 3.22 S/cm, and on the absorption coefficient in the range of 0.6–3.0 eV is observed for the nearly stoichiometric MoO3.00 and for the sub-stoichiometric MoO2.57 films, respectively, without modifying significantly the microstructure of the studied films. The presence of states within the band gap due to the lack of oxygen is the most probable mechanism for generating a change in electrical conductivity as well as optical absorption in DC-sputtered MoOx. The large tuning range of the optoelectronic properties in these films holds strong promise for their implementation in optoelectronic devices.

Responsive poly (γ-glutamic acid) fibres for biomedical applications

A novel responsive system using a protein-based biopolymer was designed to undergo structural, geometric, and chemical changes upon temperature change or solvent interaction. Poly(γ-glutamic acid) (γ-PGA) is an attractive candidate for various biomedical applications as it is naturally produced, biocompatible and enzymatically degradable. The responsive material was fabricated using an electrospun modified γ-PGA to create a sub-micron fibrous mat. By modulating the environment responsive behaviour in a controlled manner, exciting applications such as wound dressing, compression materials and self-tightening knots are envisaged.

Experimental study on polarized surface enhanced resonance Raman scattering of rhodamine 6G adsorbed on porous Al2O3 substrates

The polarization properties of surface enhanced resonance Raman scattering (SE(R)RS) of rhodamine 6G molecules, adsorbed to a hexagonally ordered gold nanostructure, are studied with the purpose to discriminate between adsorption sites with different plasmonic properties. The nanostructure is based on a self-organizing hexagonally ordered porous Al(2)O(3) substrate sputter-coated with gold. Each hexagonal subunit has D(6h) symmetry, where the symmetry center may act as an isotropic site, whereas the six narrow gaps between the individual Au hemispheres may act as hot-spots. The variation of the depolarization ratio (DPR), measured in resonance for the eight most prominent vibrational modes of the xanthene moiety, is analyzed by rotating the sample. According to theory, the DPR of the SE(R)RS signal obtained from molecules physisorbed in the isotropic sites deviates from the DPR originating from molecules physisorbed in the hot-spots in two ways: 1. The DPR associated with the isotropic sites depends differently on the rotation angle than the DPR associated with the hot-spots. 2. The DPR of the SE(R)RS signal obtained from molecules physisorbed in the isotropic sites depends on the nature of the Raman modes, whereas it for molecules physisorbed in the hot-spots is independent of the nature of the Raman modes. By applying the latter in the analysis of the polarized SE(R)RS data, we conclude that the dominating SE(R)RS signal comes from molecules adsorbed in the hot-spots. However, since the DPRs obtained for Raman modes of different symmetry are slightly different, the SE(R)RS signal must contain an additional contribution. Our analysis shows that the small mode-dependent SE(R)RS signal most likely comes from molecules adsorbed in the isotropic sites. The general result that can be derived from the present study is that by measuring the polarization properties in SE(R)RS and SERS it is possible to discriminate between adsorption sites with different plasmonic properties present in a highly symmetric nanostructure, even when the magnitude of the different contributions are highly different. The consequence of the insufficient spatial resolution with respect to a detailed mapping of the substrate often encountered in unpolarized SE(R)RS and in two-photon luminescence microscopy may thereby be circumvented.

A combination of dynamic light scattering and polarized resonance Raman scattering applied in the study of Arenicola Marina extracellular hemoglobin

Arenicola Marina extracellular hemoglobin (Hbl Hb) is considered to be a promising candidate as a blood substitute. To entangle some of the properties of extracellular giant hexagonal bilayer hemoglobin (Hbl Hb) of Arenicola Marina, we combined polarized resonance Raman scattering (532 nm excitation) with dynamic light scattering (DLS) (632.8 nm). An analysis of the depolarization ratio of selected a(2g) skeletal modes of the heme in native Hbl Hb and porcine Hb, shows that the distortion of the heme group away from its ideal fourfold symmetry is much smaller for heme groups bound in the Hbl Hb than for heme groups bound in porcine Hb. Using DLS, the average hydrodynamic diameter () of Hbl Hb was measured at pH = 5, 7, 8, 9, and 10. At pH = 5 to 7, the Hbl Hb was found in its native form with equal to 24.2 nm, while at pH = 8 and 9, a dissociation process starts to take place resulting in = 9 nm. At pH = 10, only large aggregates of fragmented Hbl Hb with larger than 1000 nm was detected, however, a comparison of the DLS results with the polarized resonance Raman scattering (RRS) revealed that the coupling between the fragments did not involve direct interaction between the heme groups, but also that the local heme environment seems to be comparable in the aggregates and in the native Hbl Hb. By comparing the unpolarized RRS results obtained for erythrocytes (RBC) with those for Hbl Hb, led us to the important conclusion that Hbl Hb is much easier photolyzed than porcine RBC.

Analysis of Reflectance and Transmittance Measurements on Absorbing and Scattering Small Samples Using a Modified Integrating Sphere Setup

The aim of the present paper is to analyze reflectance and transmittance measurements on small scattering and absorbing samples. The long term goal is to perform quantitative, spectroscopic in vivo measurements of pigments in small samples of plant material. Small samples such as small leaves constitute a special experimental challenge in cases in which the sample beam has a larger cross-sectional area than the sample. The experimental errors introduced when measuring reflectance and transmittance on small absorbing and scattering samples are investigated theoretically and experimentally by using a blue polyester sample as an appropriate test sample. The experiments are performed with either a mask or a lens setup combined with a mask inserted in the sample beam. In particular, the errors introduced in the reflectance measurements can be very large and larger than 100%. It is shown that any direct illumination of the mask must be avoided. To obtain more accurate values for the reflection coefficient it is necessary to combine the mask with a focusing lens system, adjust the mask and sample very carefully, and choose the ratio between the aperture of the mask and the beam area as large as possible. In the case of transmittance measurements, it is shown that the combination of a special sample fixture and a lens system gives rise to smaller errors compared to the errors introduced by the mask alone or the mask combined with a focusing lens system.

Two-photon luminescence microscopy of large-area gold nanostructures on templates of anodized aluminum

Using linear reflection spectroscopy and far-field two-photon luminescence (TPL) scanning optical microscopy, we characterize highly enhancing, large-area gold nanostructures formed on porous templates made by anodization of aluminum with either oxalic acid or phosphoric acid. These templates are formed by a newly developed, stepwise technique making use of protective top oxide layers facilitating continuously tunable interpore distances. The upper, porous alumina layers are subsequently removed and the remaining embossed barrier layer is used as template for the sputtered gold, where the density of gold particles covering the sample is adjusted by regulating the sputtering conditions. We observe spatially averaged field intensity enhancement (FE) factors of up to ~5.210(2) and bright spots in the TPL-images exhibiting maximum FE factors of up to approximately 1410(2) which is the largest estimated FE from any hitherto examined structures with our setup. We relate this large-area massive FE to constructive interference of surface plasmon (SP) polaritons scattered from the densely packed, randomly distributed gold particles and directly correlate this particle density with the strong and broad SP resonances as well as the magnitude of the FE factors. The average FE and the position of high enhancements in the TPL-images are dictated by the excitation wavelength, and the structures could evidently serve as versatile structures facilitating practical molecular sensing.

Periodically arranged colloidal gold nanoparticles for enhanced light harvesting in organic solar cells

Although organic solar cells show intriguing features such as low-cost, mechanical flexibility and light weight, their efficiency is still low compared to their inorganic counterparts. One way of improving their efficiency is by the use of light-trapping mechanisms from nano- or microstructures, which makes it possible to improve the light absorption and charge extraction in the device’s active layer. Here, periodically arranged colloidal gold nanoparticles are demonstrated experimentally and theoretically to improve light absorption and thus enhance the efficiency of organic solar cells. Surface-ordered gold nanoparticle arrangements are integrated at the bottom electrode of organic solar cells. The resulting optical interference and absorption effects are numerically investigated in bulk hetero-junction solar cells based on the Finite-Difference Time-Domain (FDTD) and Transfer Matrix Method (TMM) and as a function of size and periodicity of the plasmonic arrangements. In addition, light absorption enhancement in the organic active layer is investigated experimentally following integration of the nanoparticle arrangements. The latter are fabricated using a lithography-free stamping technique, creating a centimeter scaled area with nanoparticles having a defined inter-particle spacing. Our study reveals the light harvesting ability of template-assisted nanoparticle assemblies in organic solar cells. As the approach is easily scalable, it is an efficient and transferable method for large-scale, low cost device fabrication.

In Vitro Polarized Resonance Raman Study of N719 and N719-TBP in Dye Sensitized Solar Cells

The working efficiency of dye-sensitized solar cells (DSCs) depends on the long-term stability of the dye itself and on the microscopic structure of the dye-semiconductor interface. Previous experimental studies of DSCs based on ruthenium dye with bipyridine ligands (N719) adsorbed to the TiO 2 substrate applied FTIR,un-polarized Raman (RS) and un-polarized resonance Raman (RRS) spectroscopy. In the un-polarized RRS studies of N719/TiO 2 – DSCs the discussion of the adsorption of N719 was based on the rather weak carbonyl or carboxyl group stretching vibrations and on minor spectral changes of overlapping Raman modes, whereas conclusions about the dye-stability was based on the observation that fresh and aged DSCs had almost identical RRS spectra. In the present paper we address the problems mentioned above, by utilizing the unique property of Raman scattering that the polarization of the scattered light is generally different from the polarization of the laser light. When the excitation is chosen within the visible absorption band of N719 only the skeleton ring-modes in N719 are enhanced and are observed as the most intense bands in the RRS spectra. We demonstrate by experimental results on N719/TiO 2 – DSCs that by combining an analysis of the wave number dependent polarization of these modes with the small shifts observed in the visible absorption spectra of adsorbed, non-adsorbed molecules and degradation products new and more reliable information about dye stability and about the adsorption of the dye on TiO 2 can be obtained. Furthermore it is found that the polarization fluorescence anisotropy is very different for adsorbed and non-adsorbed dye molecules. This information is automatically obtained when processing the Raman data. The conclusion is that if the polarization properties of the resonance Raman spectra are analyzed instead of just analyzing the minute spectral changes of the (weaker) Raman bands the potential of RRS as an on-site tool for investigation of DSCs can be greatly improved.

In-Situ, Non-Destructive Control Of Food Quality Using Near-Infrared Raman Spectroscopy And Multivariate Analysis

In recent years there has been an increasing focus from the consumers on food quality i.e. unwanted substances such as bacteria, pesticides and drug residues, food composition and additives. This is also reflected in increasing interest for organic food products. It seems therefore appropriate to develop substance specific, nondestructive and fast measuring techniques that can be used close to the consumer, for monitoring different properties of food products. In the present paper a step is taken towards the development of an in-situ measuring technique for discriminating between different components in commercial, animal flesh products.