Quantification of fluorophore concentration and fluorescence quantum yield in tissue simulating phantoms and in vivo by fluorescence spectroscopy

The quantification of fluorophore concentration in tissue has many practical applications in the fields of pharmacokinetics, diagnosis of various diseases and the treatment of cancer. To avoid the inconveniences associated with the acquisition and processing of tissue samples (the standard method of quantitation), fast, reliable (<15% error) and non or minimally invasive methods of quantitation are desired. Measurements of fluorescence correlate well with concentration, making it a good candidate. The fluorescence signal depends on a number of factors, including the optical properties of the tissue and the local fluorophore environment (through the fluorescence quantum yield). A difference in the fluorescence quantum yield between the fluorophore in tissue and in a calibration standard, for example, would be erroneously interpreted as a change in the concentration of the fluorophore.; Two approaches were taken to estimate fluorophore concentration. In the first, fluorescence was excited and detected using a single optical fibre. In this configuration, concentration estimates were obtained from small (<1 muL) volumes with minimal dependence on the optical properties. In this first study, concentration measured on the surface and the interior of tissue-simulating phantoms could be estimated with a rootmean-square error of 10.6%. The second approach incorporated the optical properties of the tissue into two analytical models of fluorescence excitation and detection based on diffusion theory. These were compared to Monte Carlo simulations and a more complicated diffusion model that required numerical solution. If the fluorescence quantum yield was known, then the fluorophore concentration could be estimated from measurements of spatially resolved reflectance and fluorescence. The better of the two diffusion models derived in this paper yielded estimates in tissue-simulating phantoms with a root-mean-square error of 11.4%. Conversely, if the concentration was known then the fluorescence quantum yield could be estimated. The quantum yield of two fluorophores, meso-tetra-(4-sulfonatophenyl)-porphine dihydrochloride (TPPS4) and aluminum phthalocyanine tetrasulfonate (AlPcS 4) was measured accurately (0.121 +/- 0.001 cf. 0.12 and 0.59 +/- 0.03 cf. 0.58 respectively) when the concentration was estimated using an absorption measurement. (Abstract shortened by UMI.)...