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Optical diagnosis and auto-fluorescence quenching quantification of biological tissues
thesisposted on 2022-03-28, 18:38 authored by Aziz ul Rehman
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).