Spectral filtering modulation method for estimation of hemoglobin concentration and oxygenation based on a single fluorescence emission spectrum in tissue phantoms.

Hemoglobin concentration and oxygenation in tissue are important biomarkers that are useful in both research and clinical diagnostics of a wide variety of diseases such as cancer. The authors aim to develop simple ratiometric method based on the spectral filtering modulation (SFM) of fluorescence spectra to estimate the total hemoglobin concentration and oxygenation in tissue using only a single fluorescence emission spectrum, which will eliminate the need of diffuse reflectance measurements and prolonged data processing as required by most current methods, thus enabling rapid clinical measurements. The proposed method consists of two steps. In the first step, the total hemoglobin concentration is determined by comparing a ratio of fluorescence intensities at two emission wavelengths to a calibration curve. The second step is to estimate oxygen saturation by comparing a double ratio that involves three emission wavelengths to another calibration curve that is a function of oxygen saturation for known total hemoglobin concentration. Theoretical derivation shows that the ratio in the first step is linearly proportional to the total hemoglobin concentrations and the double ratio in the second step is related to both total hemoglobin concentration and hemoglobin oxygenation for the chosen fiber-optic probe geometry. Experiments on synthetic fluorescent tissue phantoms, which included hemoglobin with both constant and varying oxygenation as the absorber, polystyrene spheres as scatterers, and flavin adenine dinucleotide as the fluorophore, were carried out to validate the theoretical prediction. Tissue phantom experiments confirm that the ratio in the first step is linearly proportional to the total hemoglobin concentration and the double ratio in the second step is related to both total hemoglobin concentrations and hemoglobin oxygenation. Furthermore, the relations between the two ratios and the total hemoglobin concentration and hemoglobin oxygenation are insensitive to the scattering property of the tissue model for the chosen probe geometry. A simple two-step ratiometric method based on the SFM of fluorescence spectra is proposed to estimate the total hemoglobin concentration and oxygenation in a tissue model using only a single fluorescence emission spectrum. This method is immune to the variation in system throughput caused by inconsistent optical coupling because of its ratiometric nature. Calibration curves are insensitive to the scattering coefficient for the chosen probe geometry. Moreover, since only fluorescence intensities at a few wavelengths in a single fluorescence emission spectrum are needed in this method, the SFM method minimizes the amount of required data and reduces the data acquisition time. Finally, since this method does not use nonlinear regression, it can dramatically save computation time in data processing. The high sensitivity of the proposed method to superficial tissue volumes makes it ideal for fluorescence based oximetry and medical diagnostics in applications such as early epithelial cancer diagnosis or wherever the measured tissue volume is exposed to the outside such as in open surgery.