Advanced techniques in Raman tweezers microspectroscopy for applications in biomedicine
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This thesis investigates the use of Raman tweezers microspectroscopy to interrogate the biochemistry of single biological cells. Raman tweezers microspectroscopy is a powerful technique, which combines traditional Raman microspectroscopy and optical trapping, allowing the manipulation and environmental isolation of a biological cell of interest whilst simultaneously probing its biochemistry gleaning a wealth of pertinent information. The studies carried out in this thesis can be split into two broad categories: firstly, the exploitation of Raman tweezers microspectroscopy to study biological cells and secondly developments to the Raman tweezers microspectroscopy technique that extend its capabilities and the range of samples that can be studied. In the application of Raman tweezers, the stacking and interrogation of multiple cells is reported allowing a rapid representative Raman signal to be recorded from a small cell population with improved signal to noise. Also demonstrated is the ability of Raman spectroscopy to identify and grade the development of Human Papillomavirus induced cervical neoplasia with sensitivities of up to 96 %. These studies demonstrate the potential of Raman spectroscopy to study biological cells but it was noted that the traditional Raman tweezers system struggled to manipulate large cells thus a decoupled Raman tweezers microspectroscopy system is presented where a dual beam fibre optical trap is used to perform the trapping function and a separate Raman probe is introduced to probe the biochemical nature of the trapped cell. This development allowed the trapping and examination of very large cells whilst opening up the possibility of creating Raman maps of trapped objects. Raman tweezers microspectroscopy could potentially become an important clinical diagnostic and biological monitoring tool but is held back by the long signal integration times required due to the weak nature of Raman scattering. The final study presented in this thesis examines the potential of wavelength modulated Raman spectroscopy to improve signal to noise ratios and reduce integration times. All these studies aim to demonstrate the potential and extend the performance of Raman tweezers microspectroscopy.
Thesis, PhD Doctor of Philosophy
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