A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.

The potential of FT-IR spectroscopy for improving healthcare in sepsis - An animal model study.

Fourier Transform-Infrared (FT-IR) absorption spectroscopy has been used to investigate pathophysiological changes caused by sepsis. Sepsis has been defined as a potentially fatal organic dysfunction caused by a dysregulated host response to infection and can lead a patient to risk of death. This study used samples consisting of the blood plasma of mice which were induced to sepsis state, compared to a healthy group using FT-IR associated with attenuated total reflectance (ATR) spectroscopy. For statistical analysis, principal components analysis (PCA) and linear discriminant analysis (LDA) were applied, independently, to the second derivative spectra of both the fingerprint (900-1800 cm-1) and the high wavenumber (2800-3100 cm-1) regions. The technique efficiently differentiated the blood plasma of the two groups, sepsis and healthy mice, the analysis indicating that fatty acids and lipids in the blood samples could be an important biomarker of sepsis.

Principal components analysis of Raman spectral data for screening of Hepatitis C infection.

In the current study, Raman spectroscopy is employed for the identification of the biochemical changes taking place during the development of Hepatitis C. The Raman spectral data acquired from the human blood plasma samples of infected and healthy individuals is analysed by Principal Components Analysis and the Raman spectral markers of the Hepatitis C Virus (HCV) infection are identified. Spectral changes include those associated with nucleic acidsat720 cm-1, 1077 cm-1 1678 (CO stretching mode of dGTP of RNA), 1778 cm-1 (RNA), with proteins at 1641 cm-1(amide-I), 1721 cm-1(CC stretching of proteins) and lipids at 1738 cm-1(CO of ester group in lipids). These differences in Raman spectral features of blood plasma samples of the patients and healthy volunteers can be associated with the development of the biochemical changes during HCV infection.

Raman spectroscopy of blood plasma samples from breast cancer patients at different stages.

Raman spectroscopy was employed for the characterization of blood plasma samples from patients at different stages of breast cancer. Blood plasma samples taken from clinically diagnosed breast cancer patients were compared with healthy controls using multivariate data analysis techniques (principal components analysis - PCA) to establish Raman spectral features which can be considered spectral markers of breast cancer development. All the stages of the disease can be differentiated from normal samples. It is also found that stage 2 and 3 are biochemically similar, but can be differentiated from each other by PCA. The Raman spectral data of the stage 4 is found to be biochemically distinct, but very variable between patients. Raman spectral features associated with DNA and proteins were identified, which are exclusive to patient plasma samples. Moreover, there are several other spectral features which are strikingly different in the blood plasma samples of different stages of breast cancer. In order to further explore the potential of Raman spectroscopy as the basis of a minimally invasive screening technique for breast cancer diagnosis and staging, PCA-Factorial Discriminant Analysis (FDA) was employed to classify the Raman spectral datasets of the blood plasma samples of the breast cancer patients, according to different stages of the disease, yielding promisingly high values of sensitivity and specificity for all stages.

Raman spectral analysis for rapid screening of dengue infection.

Infection with the dengue virus is currently clinically detected according to different biomarkers in human blood plasma, commonly measured by enzyme linked immunosorbent assays, including non-structural proteins (Ns1), immunoglobulin M (IgM) and immunoglobulin G (IgG). However, there is little or no mutual correlation between the biomarkers, as demonstrated in this study by a comparison of their levels in samples from 17 patients. As an alternative, the label free, rapid screening technique, Raman spectroscopy has been used for the characterisation/diagnosis of healthy and dengue infected human blood plasma samples. In dengue positive samples, changes in specific Raman spectral bands associated with lipidic and amino acid/protein content are observed and assigned based on literature and these features can be considered as markers associated with dengue development. Based on the spectroscopic analysis of the current, albeit limited, cohort of samples, Principal Components Analysis (PCA) coupled Factorial Discriminant Analysis, yielded values of 97.95% sensitivity and 95.40% specificity for identification of dengue infection. Furthermore, in a comparison of the normal samples to the patient samples which scored low for only one of the biomarker tests, but high or medium for either or both of the other two, PCA-FDA demonstrated a sensitivity of 97.38% and specificity of 86.18%, thus providing an unambiguous screening technology.

Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices.

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in "improved viability levels" or "reduced" toxicity or cellular "resistance" compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

Retention systems for extraoral maxillofacial prosthetic implants: a critical review.

We describe the techniques available for retention of implant-supported prostheses: bar-clips, O-rings, and magnets. We present reported preferences and, although this is limited by the heterogeneity of methods used and patients studied, we hope we have identified the best retention systems for maxillofacial prosthetic implants. If practitioners know the advantages and disadvantages of each system, they can choose the most natural and comfortable prosthesis. We searched the PubMed and Scopus databases, and restricted our search to papers published 2001-13. MeSH terms used were Maxillofacial prosthesis and Craniofacial prosthesis OR Craniofacial prostheses. We found a total of 2630 papers, and after duplicates had been removed we analysed the rest and found 25 papers for review. Of these, 12 were excluded because they were case reports or non-systematic reviews. Of the remaining 13, 10 described group analyses and seemed appropriate to find practitioner's choices, as cited in the abstract (n=1611 prostheses). Three papers did not mention the type of prosthetic connection used, so were excluded. The most popular choices for different conditions were analysed, though the sites and retention systems were not specified in all 10 papers. The bar-clip system was the most used in auricular (6/10 papers) and nasal prostheses (4/10). For the orbital region, 6/10 favoured magnets. Non-osseointegrated mechanical or adhesive retention techniques are the least expensive and have no contraindications. When osseointegrated implants are possible, each facial region has a favoured system. The choice of system is influenced by two factors: standard practice and the abilities of the maxillofacial surgeon and maxillofacial prosthetist.

Raman spectroscopy for cytopathology of exfoliated cervical cells.

Cervical cancer is the fourth most common cancer affecting women worldwide but mortality can be decreased by early detection of pre-malignant lesions. The Pap smear test is the most commonly used method in cervical cancer screening programmes. Although specificity is high for this test, it is widely acknowledged that sensitivity can be poor mainly due to the subjective nature of the test. There is a need for new objective tests for the early detection of pre-malignant cervical lesions. Over the past two decades, Raman spectroscopy has emerged as a promising new technology for cancer screening and diagnosis. The aim of this study was to evaluate the potential of Raman spectroscopy for cervical cancer screening using both Cervical Intraepithelial Neoplasia (CIN) and Squamous Intraepithelial Lesion (SIL) classification terminology. ThinPrep® Pap samples were recruited from a cervical screening population. Raman spectra were recorded from single cell nuclei and subjected to multivariate statistical analysis. Normal and abnormal ThinPrep® samples were discriminated based on the biochemical fingerprint of the cells using Principal Component Analysis (PCA). Principal Component Analysis - Linear Discriminant Analysis (PCA-LDA) was employed to build classification models based on either CIN or SIL terminology. This study has shown that Raman spectroscopy can be successfully applied to the study of routine cervical cytology samples from a cervical screening programme and that the use of CIN terminology resulted in improved sensitivity for high grade cases.

Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices.

The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in "reduced" toxicity or cellular "resistance" to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug and assay in the 3D environment. The effect was noted to be higher after shorter exposure periods and should be accounted for in in vitro 2D and 3D culture environment comparisons.

Cellular discrimination using in vitro Raman micro spectroscopy: the role of the nucleolus.

Raman micro spectroscopy has attracted considerable attention over the last few years to explore its possible clinical applications as a non-invasive powerful label-free in vitro screening tool in cancer diagnosis and monitoring, subcellular analysis of biochemical processes, drug uptake, mode of action and mechanisms of interaction as well as toxicity of, for example, chemotherapeutic agents. However, in order to evaluate accurately the potential of Raman micro spectroscopy for such applications it is essential to optimise measurement and data processing protocols associated with subcellular analysis. To this end, in vitro differentiation of cell lines is a basic proof of concept for the potential of the technique, and although many studies have indicated successful differentiation based on Raman micro spectroscopy, it is important, as the measurement and processing techniques are improved, to establish the biochemical and subcellular basis of that discrimination. In this study, Raman micro spectroscopy is used to compare and differentiate normal and cancer cells from human lung origin, A549 adenocarcinoma cell line, Calu-1 epidermoid non-small-cell and BEAS-2B normal immortalized bronchial epithelium cell line. Spectra were taken from the three subcellular compartments, cytoplasm, nucleus and nucleolus and Principal Components Analysis was used to compare the spectral profiles between the cell lines and, coupled to Linear Discriminant Analysis, to explore the optimum sensitivity and specificity of discrimination. To support the analysis, Raman micro spectroscopy was coupled with Flow Cytometry, Confocal Laser Scanning Microscopy and Atomic Force Microscopy. While all subcellular regions can be employed to differentiate the normal and cancer cell lines, optimum discrimination sensitivity and specificity is achieved using the spectra from the nucleolar region alone. Notably, only the nucleolar spectral profiles differentiate the two cancer cell lines. The results point to the importance of the nucleolar regions in diagnostic applications of Raman microscopy as well as further applications in subcellular analysis of cytological processes.

Raman micro spectroscopy for in vitro drug screening: subcellular localisation and interactions of doxorubicin.

Vibrational spectroscopy, including Raman micro spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman micro spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents. In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. In this study paper, Raman micro spectroscopy coupled with confocal microscopy were used for the localization and tracking of the commercially available drug, doxorubicin (DOX), in the intracellular environment of the lung cancer cell line, A549. Cytotoxicity assays were employed to establish clinically relevant drug doses for 24 h exposure, and Confocal Laser Scanning Fluorescence Microscopy was conducted in parallel with Raman micro spectroscopy profiling to confirm the drug internalisation and localisation. Multivariate statistical analysis, consisting of PCA (principal components analysis) was used to highlight doxorubicin interaction with cancer cells and spectral variations due to its effects before and after DOX spectral features subtraction from nuclear and nucleolar spectra, were compared to non-exposed control spectra. Results show that Raman micro spectroscopy is not only able to detect doxorubicin inside cells and profile its specific subcellular localisation, but, it is also capable of elucidating the local biomolecular changes elicited by the drug, differentiating the responses in different sub cellular regions. Further analysis clearly demonstrates the early apoptotic effect in the nuclear regions and the initial responses of cells to this death process, demonstrating the potential of the technique to monitor the mechanisms of action and response on a molecular level, with subcellular resolution.

Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models.

Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorimetric dye-based cytotoxic assays. Using the well established Alamar blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells.

Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells.

There is much evidence supporting the existence of bystander effects in cells that were never exposed to radiation. Directly irradiated cells and bystander cells can communicate with each other using gap junctional intercellular communication or by releasing soluble factors into the surrounding medium. Exosomes and microvesicles are also known to mediate communication between cells. The main aim of this study is to establish whether exosomes and microvesicles are involved in radiation induced bystander signaling. Human keratinocytes, HaCaT cells, were irradiated (0.005, 0.05 and 0.5 Gy) using γ rays produced from a cobalt 60 teletherapy unit. After irradiation, the cells were incubated for 1 h and the irradiated cell conditioned medium (ICCM) was harvested. Exosomes were isolated from the ICCM using ultracentrifugation. Exosomes were characterized using light scattering analysis (LSA) and scanning transmission electron microscopy (STEM). Cytotoxicity and reactive oxygen species assays and real time calcium imaging were performed either with ICCM from which exosomes and microvesicles were removed or with the exosome fraction resuspended in cell culture media. The characterization data showed a particle size distribution indicative of both exosomes (30-100 nm) and microvesicles (>100 nm) and the light scattering analysis showed increased concentration of both exosomes and microvesicles with increasing dose. Western blotting confirmed the presence of an exosomal protein marker, TSG 101. Treatment of unirradiated cells with ICCM in which exosomes and microvesicles were removed resulted in abrogation of ICCM induced effects such as reduction in viability, calcium influx and production of reactive oxygen species. Addition of exosomes to fresh media produced similar effects to complete ICCM. These results suggest a role for exosomes and microvesicles in radiation induced bystander signaling.

Discrimination of cathinone regioisomers, sold as 'legal highs', by Raman spectroscopy.

The discrimination of a cross section of cathinone regioisomers, sold as 'legal highs', using Raman spectroscopy, is reported here. Mephedrone and flephedrone were identified in 'legal high' products sold in Irish head shops, and their 2, 3 and 4-isomers were synthesized as reference standards. The 3,4-methylenedioxy substituted cathinones, methylone, butylone and methylenedioxypyrovalerone (MDPV), were also identified in 'legal highs' and their 2,3-isomers were synthesized for comparison. In addition, alpha- and beta-naphyrone were synthesized. Raman spectra of all the isomers were obtained using far-red excitation (785 nm) and it was found possible to discriminate the isomers of each substituted cathinone. In addition, Raman spectra were also recorded for a number of head shop products and, by comparison with the reference standards, correct isomer assignment for 4-mephedrone, 3-flephedrone, 3,4-methylone, 3,4-butylone, 3,4-MDPV, alpha-naphyrone and beta-naphyrone was achieved, thus providing a non-destructive, high-throughput and minimal sample preparation technique for the discrimination of such drug isomers.

Raman spectroscopic mapping for the analysis of solar radiation induced skin damage.

The effects of simulated solar irradiation of an artificial skin model have been examined using Raman spectroscopy and the results are compared with cytotoxicological and histological profiling. Samples exposed for times varying between 30 minutes and 240 minutes were incubated post exposure for a period of 96 hours. The cytotoxicological response as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay demonstrated a ~50% loss of viability of the artificial tissue after 120 minutes exposure. Histological staining of tissue sections showed considerable loss of cellular content in the epidermal layer at this endpoint. Raman spectroscopic mapping of tissue sections, coupled with K-means cluster analysis (KMCA) clearly identified the dermal and stratum corneum layers and differentiated further substructures of the epidermis. Post irradiation, a significant loss of DNA features in the basal layer was apparent in the results of the KMCA. Principal Components Analysis (PCA) of layers identified by the KMCA post exposure compared with controls indicated a significant increase in the lipidic signatures of the stratum corneum. In the dermal layer, little photodamage was observed, but a similar increase in lipidic signatures in the basal layer was accompanied by a decrease in DNA and protein contributions. The spectral profiles of the photodamage to the basal layer as identified by PCA are consistent over the exposure periods of 30-240 minutes, but an examination of the evolution of features associated with specific biochemical components indicated DNA damage and loss of lipidic signatures at the early exposure times, whereas changes in protein signatures appeared to evolve over longer periods. In comparison to the cytotoxicological responses, the study demonstrates that Raman spectroscopy can identify biochemical changes as a result of solar exposure at time points significantly earlier than changes in tissue viability are observed.

Understanding the molecular information contained in principal component analysis of vibrational spectra of biological systems.

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 similarly 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 becomes 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.

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy.

Three dimensional collagen gels have been used as matrices for the imaging of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. The lateral resolution of the system has been estimated to be <1.5 µm making it possible to access the subcellular organization. Using K-means clustering analysis, it is shown that the different subcellular compartments of individual cells can be identified and differentiated. The biochemical specificity of the information contained in the Raman spectra allows the visualization of differences in the molecular signature of the different sub-cellular structures. Furthermore, to enhance the chemical information obtained from the spectra, principal component analysis has been employed, allowing the identification of spectral windows with a high variability. The comparison between the loadings calculated and spectra from pure biochemical compounds enables the correlation of the variations observed with the molecular content of the different cellular compartments.

Three dimensional collagen gels as a cell culture matrix for the study of live cells by Raman spectroscopy.

Three dimensional collagen gels are evaluated as matrices for the study of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. It is demonstrated, using the Alamar Blue assay, that both cell models exhibit enhanced viability in collagen matrices compared to quartz substrates, commonly used for Raman spectroscopy. Using principal component analysis, it is shown that the Raman spectral analysis of cells in collagen matrices is minimally contaminated by substrate contributions and the cell to cell spectral variations are greatly reduced compared to those measured on quartz substrates. Furthermore, the spectral measurements are seen to have little contribution from the cell culture medium, implying that cultures can be kept viable over prolonged measurement or mapping procedures.

An investigation of the RWPE prostate derived family of cell lines using FTIR spectroscopy.

Interest in developing robust, quicker and easier diagnostic tests for cancer has lead to an increased use of Fourier transform infrared (FTIR) spectroscopy to meet that need. In this study we present the use of different experimental modes of infrared spectroscopy to investigate the RWPE human prostate epithelial cell line family which are derived from the same source but differ in their mode of transformation and their mode of invasive phenotype. Importantly, analysis of the infrared spectra obtained using different experimental modes of infrared spectroscopy produces similar results. The RWPE family of cell lines can be separated into groups based upon the method of cell transformation rather than the resulting invasiveness/aggressiveness of the cell line. The study also demonstrates the possibility of using a genetic algorithm as a possible standardised pre-processing step and raises the important question of the usefulness of cell lines to create a biochemical model of prostate cancer progression.

Spectroscopic and chemometric approaches to radiobiological analyses.

Vibrational spectroscopy is an attractive modality for the analysis of biological samples, providing a complete non-invasive acquisition of the biochemical fingerprint of the sample. It has been demonstrated that this data provides the means to assay multiple functional responses of a biological system at a spatial resolution as low as a micron within the sample. As the interaction of ionizing radiation with biological systems involves chemical reactions between the products of radiation-induced damage and various structural and functional units within the cell, the vibrational spectroscopic modalities have received attention as potential measurement platforms for the in situ examination of the chemistry of biological species in radiobiology. This presents challenges in relation to sample preparation and the construction of suitable analytical methodologies. In this work protocols for sample preparation and approaches to multivariate analysis of vibrational spectra in radiobiological analysis are detailed and the utility of the methodology in analyzing the evolution of biochemical responses to radiobiological damage are highlighted.