Infrared spectroscopic cytology is potentially a powerful clinical tool. However, in order for it to be successful, practitioners must be able to extract reliably a pure absorption spectrum from a measured spectrum that often contains many confounding factors. The most intractable problem to date is the, so called, dispersion artefact which most prominently manifests itself as a sharp decrease in absorbance on the high wavenumber side of the amide I band in the measured spectrum, exhibiting a derivative-like line shape. In this paper we use synchrotron radiation FTIR micro-spectroscopy to record spectra of mono-dispersed poly(methyl methacrylate) (PMMA) spheres of systematically varying size and demonstrate that the spectral distortions in the data can be understood in terms of resonant Mie scattering. A full understanding of this effect will enable us to develop strategies for deconvolving the scattering contribution and recovering the pure absorption spectrum, thus removing one of the last technological barriers to the development of clinical spectroscopic cytology.

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The dependence of the Surface Enhanced Raman Scattering (SERS) by gold nanoparticles on their shape is examined using the organic dye, rhodamine 6G (R6G) as probe molecule. SERS has been explored extensively for applications in sensing and imaging, but the design and optimisation of efficient substrates is still challenging. In order to understand and optimise the SERS process in nanoparticles, gold nanospheres and their aggregates, nanotriangles, and nanostars of similar dimensions were synthesised and characterised according to their average size, zeta potential and UV/visible absorption. SERS from R6G was negligible for unaggregated nanospheres at 532 nm, close to the maximum of the surface plasmon resonance (SPR) at 560 nm. Upon aggregation of the nanospheres, the SPR shifts to ~660 nm, attributable to local surface plasmon "hotspots" between the spheres, and the SERS signal of R6G is significantly increased, at 785 nm. In monodisperse gold nanotriangles, the SPR is located at ~800 nm, and significant SERS of R6G is observed using 785 nm as source, as is the case for gold nanostars, which exhibit a double SPR with maxima at ~600 nm and ~785 nm, attributable to the core sphere and vertices of the structures, respectively. In suspensions of equal nanoparticle and dye concentration, the SERS effect increases as nanospheres

Abstract:K-means clustering followed by Principal Component Analysis (PCA) is employed to analyse Raman spectroscopic maps of single biological cells. K-means clustering successfully identifies regions of cellular cytoplasm, nucleus and nucleoli, but the mean spectra do not differentiate their biochemical composition. The loadings of the principal components identified by PCA shed further light on the spectral basis for differentiation but they are complex and, as the number of spectra per cluster is imbalanced, particularly in the case of the nucleoli, the loadings under-represent the basis for differentiation of some cellular regions. Analysis of pure bio-molecules, both structurally and spectrally distinct, in the case of histone, ceramide and RNA, and similar in the case of the proteins albumin, collagen and histone, show the relative strong representation of spectrally sharp features in the spectral loadings, and the systematic variation of the loadings as one cluster becomes reduced in number. The more complex cellular environment is simulated by weighted sums of spectra, illustrating that although the loading become increasingly complex; their origin in a weighted sum of the constituent molecular components is still evident. Returning to the cellular analysis, the number of spectra per cluster is artificially balanced by increasing the weighting of the spectra of smaller number clusters. While it renders the PCA loading more complex for the three-way analysis, a pair wise analysis illustrates clear differences between the identified subcellular regions, and notably the molecular differences between nuclear and nucleoli regions are elucidated. Overall, the study demonstrates how appropriate consideration of the data available can improve the understanding of the information delivered by PCA.

The study of the interaction of anticancer drugs with mammalian cells in vitro is important to elucidate the mechanisms of action of the drug on its biological targets. In this context, Raman spectroscopy is a potential candidate for high throughput, non-invasive analysis. To explore this potential, the interaction of cis-diamminedichloroplatinum(II) (cisplatin) with a human lung adenocarcinoma cell line (A549) was investigated using Raman microspectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC 50 value of 1.2 AE 0.2 mM. To further confirm the spectral results, Raman spectra were also acquired from DNA extracted from A549 cells exposed to cisplatin and from unexposed controls. Partial least squares (PLS) multivariate regression and PLS Jackknifing were employed to highlight spectral regions which varied in a statistically significant manner with exposure to cisplatin and with the resultant changes in cellular physiology measured by the MTT assay. The results demonstrate the potential of the cellular Raman spectrum to noninvasively elucidate spectral changes that have their origin either in the biochemical interaction of external agents with the cell or its physiological response, allowing the prediction of the cellular response and the identification of the origin of the chemotherapeutic response at a molecular level in the cell.