Vibrational spectroscopic analysis of control and altered biological targets from mm to nm length scale
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The aim of this thesis was to develop correlative applications using far-field FTIR (FFIR), Attenuated Total Reflectance FTIR, nano-FTIR, and Optical Photothermal IR spectroscopy (O-PTIR) to obtain the spectral signatures of control and altered biological samples and investigate their responses to modifications at different length scales (mm to nm). The spatial resolution of FTIR microscopy is restricted by the diffraction limit, approximately the wavelength of the imaging light. The diffraction limit to spatial resolution can be broken using nano-FTIR at 20 nm spatial resolution, with either a synchrotron light source or broadband nano-FT-IR quantum cascade laser source. O- PTIR also overcomes the IR wavelength-dependent resolution; its spatial resolution is determined by the wavelength of the visible probe laser, typically about ½ µm, and independent of the IR wavelength. In the first part of this thesis, the structure of intact bovine tendons and fibrils were examined, exploiting the effects of polarized IR light with FFIR and near-field IR techniques. FFIR with FPA data yield spectral information only from intact tendons, while O-PTIR data yield information from both tendons and fibrils (~500 nm). Only nano-FTIR delivers spectra from sub-fibrils with diameters ~100 nm. Subsequently, mechanically-damaged fibrils from load-bearing and positional bovine forelimb tendons are studied, revealing positional tendons' higher susceptibility to molecular-level structural changes. In the second part of the thesis, nano-FTIR spectroscopy was used to characterize the fungi cell wall of Aspergillus nidulans and Saccharomyces cerevisiae. Nano-FTIR emerges as the best non-destructive technique for nanoscale samples, exemplified by the study of cell walls with a thickness of ~100 nm. Each spectrum from different mutants displays characteristic changes, offering insights into gene deletions' impact on biochemical composition. Overall, in this thesis it has been possible to use the broad applicability of IR spectroscopy for studying different biological targets using standard and novel techniques. The findings have implications for multidisciplinary research, including bioengineering, agriculture, and pharmaceuticals, aiding in the development of new biological materials and antifungal drugs.