Raman Spectroscopy of Human Lens Epithelial Cells Exposed to a Low-Dose Range of Ionizing Radiation

Abstract

Recent studies indicate that ionizing radiation induced opacification in the lens of the eye occurs at lower doses (< 2 Gy) than past protection guidelines had assumed. Research is currently focused on identifying early signs of the lens degradation that leads to cataract formation, and in developing non-invasive assays capable of detecting low dose exposures to the lens of the eye. Raman spectroscopy (RS) is a non-invasive, vibrational spectroscopic technique based on the inelastic scattering of light by molecular vibrations. It is capable of providing information on the molecular makeup of biological samples that can be used for classification purposes. This work focuses on the application of RS combined with multivariate statistical analysis to detect radiation induced changes in vitro within human lens epithelial (HLE) cells exposed to a broad dose-range (0.01-5 Gy). The development of a new Raman microscope which will increase data acquisition throughput is also discussed. I would like to begin by thanking all members of the Carleton Biophotonics Research Group (CBRG), both past and present. First and foremost, I would like to express my heartfelt gratitude to my supervisor, Dr. Sangeeta Murugkar, for providing invaluable advice and guidance, for keeping me on track throughout my work, and without whom this research would not have happened. I would like to thank my predecessor, Hamid Moradi, who, with the help of Abrar Ahmad and Dean Sheperdson, built the original instrument on which my research was performed, and who showed me every detail about how it works, how to align it, and how to use it. Hamid provided me a solid foundation of both knowledge and skill in this field for which I will always be grateful. Dr. Balazs Nyiri of the Ottawa Hospital is another member of the CBRG who provided invaluable advice and opinions critical to the experiment as a whole. I would like to thank Abrar Ahmad, Christopher Dedek, and Achint Kumar, whose work was also instrumental in performing this experiment. Abrar wrote the analysis code on which I based my own and which I always used to cross-check results. Chris figured out how to automate the stage in coordination with the spectroscopic software, which simplified things immensely. Achint was my partner in data acquisition and initial analyses. I am grateful for his willingness to take the late shift on most of our day long acquisitions. I am also grateful for the work done by new team member Ben Hansson, who determined which laser and optical fibers would be best for the new Raman microscope, and Aditya Babu, who assisted me in aligning and testing the new Raman microscope, as well as in building the new brightfield illumination path. From Health Canada, I would like to sincerely thank Dr. Vinita Chauhan and Dr. Sami Qutob for their invaluable contributions. Dr. Chauhan conceived of the need for this work, and was responsible for growing, irradiating, and transporting ii the cells. Dr. Qutob provided the clonogenic assay data and helped with growing and transporting cell...

Development of a flow cell based Raman spectroscopy technique to overcome photodegradation in human blood

“…The use of a water immersion objective instead of an air objective to focus the laser on the blood lysate sample enables better matching of the refractive indices across the blood/quartz and water interface and thus results in higher Raman signal collection [23]. The multimode laser beam is focused to a spot whose lateral diameter and depth of focus was measured to be 12 µm and 25 µm respectively [24]. The lateral diameter of the laser spot was empirically determined from a calibrated bright field image of the focused laser spot while the depth of focus was determined by acquiring Raman spectra from various heights at the surface of a polystyrene dish [25].…”