Quantification Of Protoporphyrin IX(PpIX) Fluorescence In Tissue

Biomedical photonics technology is playing an increasingly important role in the diagnosis and treatment of diseases.Photodynamic diagnosis(PDD)and photodynamic therapy(PDT),which are based on the combination of photosensitizer and illumination,are one of examples of such technology.PDD and PDT have the advantages of noninvasive or minimally invasive,high specificity,safety and less side effects.At present,a variety of photosensitizers and precursors have been approved in China and abroad for the diagnosis and treatment of a wide range of benign and malignant diseases in various parts of the body.5-aminolevulinic acid(ALA)and its ester derivatives are the most widely used precursors worldwide.They are often called "the second generation photosensitizer".ALA can be transformed into protoporphyrin IX(PpIX)in cells.PpIX is a kind of porphyrin processing strong photosensitivity.When exposed to ultraviolet light it can produce characteristic red fluorescence,which can be used for visualization and differential diagnosis of lesions.When exposed to red and blue light it can produce singlet oxygen,which possesses unique therapeutic power.ALA/PpIX-based PDD and PDT are an effective and practical technique for in situ diagnosis and treatment.As in this process PpIX is an indirect product,its dose is strongly affected by many variations in individual patients.Therefore,in the era of precision medicine,real-time and in vivo quantitative detection of PpIX is of great significance.However,in the development and application of PDD and PDT,the quantitative detection of photosensitizer is still a technical problem unsolved.This has also become the bottleneck in the transition from experience-based method to a precision modality for PDD and PDT that ultimately limits the preciseness of PDD and PDT.Early studies show that the quantitative detection of PpIX fluorescence might provide a useful means for quantitative detection of PpIX in biological tissues,but the commonly used fluorescence detection equipment and method cannot meet the needs of fluorescence quantification.In this thesis,the quantitative detection methods of PpIX fluorescence in tissues were systematically studied.First,the concept,significance and research progress of fluorescence quantitative detection of photosensitizers in tissues were described.The fluorescence quantitative detection methods of photosensitizers in tissues reported in literature were reviewed.Based on the basic principles of tissue optics,the main factors affecting the quantitative fluorescence detection of photosensitizers were explored.The dual band fluorescence of tissue diffuse reflectance spectrum in excitation and emission bands was examined.Under the consideration of the accuracy of measurement,the feasibility of clinical application,and the needs of experimental measurement equipment,a method for quantitative detection of PpIX in tissues was designed,developed and evaluated.The research work of this thesis include:1.Design and construct a fluorescence spectrum detection system.The system was used for contact point measurement and non-contact surface measurement.The software of data analysis for fluorescence spectrum quantification was written based on Python language.The measurement principle and error source of the detection system were analyzed,the method of error calibration of the detection system was given and the performance of the system was evaluated in various tissue phantoms.2.Develop a dual band tissue fluorescence spectrum calibration method.Monte Carlo method was used to simulate tissue fluorescence and diffuse reflectance to verify the validity of dual band tissue fluorescence spectrum calibration algorithm based on tissue excitation and emission band diffuse reflectance spectrum.The fluorescence of PpIX in optical simulation solution of skin,muscle and soft tissue was measured,the calibration algorithm of PpIX fluorescence spectrum was established and calibration results were evaluated.3.Develop the calibration method for tissue PpIX fluorescence spectrum in vivo.Using fluorescence spectrum detection system and dual band tissue fluorescence spectrum calibration method,the PpIX fluorescence of in subcutaneous tumor tissue was quantitatively detected in vivo,and the concentration of photosensitizer in tissue was obtained,and the separation and quantitative detection method of PpIX by high performance liquid chromatography(HPLC)combined with ultraviolet absorption spectrometry was established.The concentration of PpIX in tumor tissue extract was quantitatively detected and compared with in vivo quantitative results obtained from the detection system.4.Analyze the influence of PpIX concentration distribution in tissues on fluorescence detection.Based on the characteristics of dual wavelength excitation light source of fluorescence detection system,the relationship between the intensity ratio of PpIX fluorescence characteristic peak and the thickness of tissue was analyzed by combining Monte Carlo simulation with tissue simulation liquid experiment.Three models of PpIX spatial distribution,i.e.uniform distribution,surface distribution and deep distribution,were constructed to analyze the influence of PpIX concentration distribution in tissue.The ratio of fluorescence characteristic peak intensity was used for analyzing the concentration distribution of PpIX in tissues.