Optical diagnosis and auto-fluorescence quenching quantification of biological tissues

This thesis contributes to the development of non-invasive optical techniques based on light absorption, scattering, and fluorescence for photo-diagnostic and Photo-dynamic Therapy (PDT) of biological tissues. The first part of the thesis is devoted to the development of highly diffusive tissue body phantoms for optical parameter measurements aiming differentiation of healthy and diseased tissues. The key results are theoretical and experimental investigations of photon transport in biological tissues. The diffuse reflectance Rd and diffuse transmittance Td of tissue body phantoms were measured using Double Integrating Sphere system. The optical parameters, absorption coefficient µa and reduce scattering coefficient ´ µs were calculated employing Inverse Adding-Doubling method from the measured values of diffuse reflectance Rd and diffuse transmittance Td. This part also includes breast cancerous-tissue differentiation from normal tissue on the basis of Raman Scattering, Polarization and Confocal Fluorescence Imaging. The second part of the research is devoted to the fluorescence diagnostics of biological tissues and cells. The Programmable Integrating Sphere Light (PISAL) source was designed, built and retro-fitted in Laser Scanning Leica-DMIRB Microscope for wide-field fluorescence microscopy of BV2 cancerous cell line. The in-vitro fluorescence chemical quenching quantification of the native fluorophore, free and bound Reduced Nicotinamide Adenine Dinucleotide (NADH) was performed. Key results of fluorescence quenching quantification confirm, that Carbonyl Cyanide-P-Trifluoro-Methoxy Phenyl Hydrazone (FCCP) selectively quenches the fluorescence of free and bound-NADH in plated and suspended He-La cells. The auto-fluorescence quenching quantification of NADH/ NAD(P)H with FCCP has validated the results of unsupervised unmixing in He-La cell using label-free optical method of Hyperspectral Auto-Fluorescence Imaging. The combination of Hyperspectral Auto-Fluorescence Imaging and unsupervised unmixing technique will be useful for tissue diagnostic for monitoring of Photo-dynamic Therapy using PISAL light source and Single Channel Analysis(SCA).