Selection of Preprocessing Methodology for Multivariate Regression of Cellular FTIR and Raman Spectra in Radiobiological Analyses

Vibrational spectra of biological species suffer from the influence of many extraneous interfering factors that require removal through preprocessing before analysis. The present study was conducted to optimise the preprocessing methodology and variable subset selection during regression of and confocal Raman microspectroscopy (CRM) and Fourier Transform Infrared microspectroscopy (FTIRM) spectra against ionizing radiation dose. Skin cells were γ-irradiated in-vitro and their Raman and FTIRM spectra were used to retrospectively predict the radiation dose using linear and nonlinear partial least squares (PLS) regression algorithms in addition to support vector regression (SVR). The optimal preprocessing methodology (which comprised combinations of spectral filtering, baseline subtraction, scaling and normalization options) was selected using a genetic algorithm (GA) with the root mean squared error of prediction (RMSEP) used as the fitness criterion for selection of the preprocessing chromosome (where this was calculated on an independent set of test spectra randomly selected from the dataset on each pass of the algorithm). The results indicated that GA selection of the optimal preprocessing methodology substantially improved the predictive capacity of the regression algorithms over baseline methodologies, although the optimal preprocessing chromosomes were similar for various regression algorithms, suggesting an optimal preprocessing methodology for radiobiological analyses with biospectroscopy. Feature selection of both FTIRM and CRM spectra using genetic algorithms and multivariate regression provided further decreases in RMSEP, but only with non-linear multivariate regression algorithms

Integration of New Biological and Physical Retrospective Dosimetry Methods Into EU Emergency Response Plans – Joint RENEB and EURADOS Inter-Laboratory Comparisons

RENEB, \u27Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,\u27 is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation

Optimisation of Raman Spectral Processing for Classification of Radiotherapeutic Toxicity

Severe radiation toxicity can continue years after the completion of radiotherapy for prostate cancer patients. Currently, it is impossible to predict before treatment which patients will experience these long-term side effects. New approaches based on vibrational spectroscopy have advantages over lymphocyte and genomic assays in terms of minimal sample preparation, speed and cost. A high throughput method has been developed to measure Raman spectra from liquid plasma in a cover glass bottomed 96 well plate. However, the Raman spectra can show contributions from glass and water. The current study aims to optimise pre-processing steps to improve classification performance

Raman Micro Spectroscopy Study of the Interaction of Vincristine with A549 Cells Supported by Expression Analysis of bcl-2 Protein

Understanding the interaction of anticancer drugs with model cell lines is important to elucidate the mode of action of these drugs as well as to develop cost effective and rapid screening methods. Raman spectroscopy has been demonstrated to be a valuable technique for high throughput, noninvasive analysis. The interaction of vincristine with a human lung adenocarcinoma cell line (A549)was investigated using Raman micro spectroscopy. The results were correlated with parallel measurements from the MTT cytotoxicity assay, which yielded an IC50 value of 0.10 ± 0.03 μM. The Raman spectral data acquired from vincristine treated A549 cells was analysed to understand its interaction with the nucleus in the cell and elucidate DNA intercalation. The dose dependent spectral changes in the nucleus are analysed by PLS-Jack knifing for the identification of the more significant changes associated with the mode of action of the drug. Results are correlated with a similar dose dependent expression analysis of the bcl-2 protein, an anti-apoptotic protein associated with DNA damage, in the vincristine treated A549 cells using flow cytometry. The results indicate the co-existence of two modes of action, microtubule binding at low doses and DNA intercalation at high doses

Effect of Pre-Analytical Variables on Raman and FTIR Spectral Content of Lymphocytes

The use of Fourier transform infrared (FTIR) and Raman spectroscopy (RS) for the analysis of lymphocytes in clinical applications is increasing in the field of biomedicine. The pre-analytical phase, which is the most vulnerable stage of the testing process, is where most errors and sample variance occur; however, it is unclear how pre-analytical variables affect the FTIR and Raman spectra of lymphocytes. In this study, we evaluated how pre-analytical procedures undertaken before spectroscopic analysis influence the spectral integrity of lymphocytes purified from the peripheral blood of male volunteers (n = 3). Pre-analytical variables investigated were associated with (i) sample preparation, (blood collection systems, anticoagulant, needle gauges), (ii) sample storage (fresh or frozen), and (iii) sample processing (inter-operator variability, time to lymphocyte isolation). Although many of these procedural pre-analytical variables did not alter the spectral signature of the lymphocytes, evidence of spectral effects due to the freeze–thaw cycle, in vitro culture inter-operator variability and the time to lymphocyte isolation was observed. Although FTIR and RS possess clinical potential, their translation into a clinical environment is impeded by a lack of standardisation and harmonisation of protocols related to the preparation, storage, and processing of samples, which hinders uniform, accurate, and reproducible analysis. Therefore, further development of protocols is required to successfully integrate these techniques into current clinical workflows

Exploring Technology Enhanced Instruction and Assessment in the Advanced Physics Laboratory

The development of a strategic, thoughtful and reflective approach to the undertaking of experimental work is key to the development of physicists and physical scientists. This project undertook to remodel senior physics laboratories to adapt to changing skillsets required in the workplace and to instil the graduate attributes necessary for flexible employment in physics and related disciplines. The objective of this project was to foster an enquiry-based model that has been shown both to help engage the students with their subject and develop habits of experimental approach appropriate to physical scientists. The project used e-assessment methods and electronic documentation of student experimental planning, reflection and data recording, while adjusting laboratory instructions and resources. These adjustments included minimising experimental procedures such that this created a less restrictive and more free-form experimental experience, challenging the students to plan prior to experimental work and reflect afterwards. A number of evaluation techniques were used to measure the impact of these changes, including an anonymised on-line survey using the University of Colorado E-CLASS (Colorado Learning Attitudes about Science Survey for Experimental Physics) survey and peer evaluation of experimental reports. The E-CLASS survey was also taken by School of Physics staff to provide a measure of the expert view. The success of this new approach to laboratory instruction is seen in the general alignment between student views of issues of importance in experimental approach. This innovative approach to laboratory instruction will continue to be evaluated and refined for future student cohorts

Discrimination of immune cell activation using Ramanmicro-spectroscopy in anin-vitro & ex-vivo model

Activation and proliferation of immune cells such as lymphocytes and monocytes are appropriate inflammatory responses to invading pathogens and are key to overcoming an infection. In contrast, uncontrolled and prolonged activation of these cellular signalling pathways can be deleterious to the body and result in the development of autoimmune conditions. The understanding of cellular activatory status therefore plays a significant role in disease diagnosis and progression. Conventional automated approaches such as enzyme linked immunosorbent assays (ELISA) and immune-labelling techniques are time-consuming and expensive, relying on a commercially available and specific antibody to identify cell activation. Developing a label-free method for assessing molecular changes would therefore offer a quick and cost-efficient alternative in biomedical research. Here Raman spectroscopy is presented as an effective spectroscopic method for the identification of activated immune cells using both cell lines and primary cells (including purified monocyte and lymphocyte subgroups and mixed peripheral blood mononuclear cell (PBMC) populations) obtained from healthy donors. All cell lines and primary cells were exposed to different stimulants and cellular responses confirmed by flow cytometry or ELISA. Machine learning models of cell discrimination using Raman spectra were developed and compared to reference flow-cytometry, with spectral discrimination levels comparing favourably with the reference method. Spectral signatures of molecular expression after activation were also extracted with results demonstrating alignment with expected profiles. High performance classification models constructed in these in-vitro and ex-vivo studies enabled identification of the spectroscopic discrimination of immune cell subtypes in their resting and activated state. Further spectral fitting analysis identified a number of potential spectral biomarkers that elucidate the spectral classification

Towards plasmon mapping of SERS-active Ag dewetted nanostructures using SPELS

Thermal dewetting of silver thin flm can lead to SERS-active Ag nanoparticles. Here, we report our progress towards using scanning probe energy loss spectroscopy (SPELS) to map the plasmonic behaviour of SERS-active Ag nanoparticles (NP) by investigating NPs produced through the dewetting study of Ag thin flms on SiO2/Si and Ti/SiO2/Si substrates. The nanoparticles size and spatial distribution were controlled by the deposition and thermal annealing parameters. The results of preliminary SPELS measurements of these structures, alongside SERS data show that there is a correlation between the Raman enhancement and the nanoparticle size and interparticle spacing

Vibrational spectroscopy in sensing radiobiological effects: analyses of targeted and non-targeted effects in human keratinocytes

Modern models of radiobiological effects include mechanisms of damage initiation, sensing and repair, for those cells that directly absorb ionizing radiation as well as those that experience molecular signals from directly irradiated cells. In the former case, the effects are termed targeted effects while, in the latter, non-targeted effects. It has emerged that phenomena occur at low doses below 1Gy in directly irradiated cells which are associated with cell-cycle dependent mechanisms of DNA damage sensing and repair. Likewise in non-targeted bystander irradiated cells the effect saturates at 0.5Gy. Both effects at these doses challenge the limits of detection of vibrational spectroscopy. In this paper, a study of the sensing of both targeted and non-targeted effects in HaCaT human keratinocytes irradiated with gamma-ray photons is conducted with vibrational spectroscopy