Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.
We report preliminary clinical results of autofluorescence imaging of malignant and benign skin lesions, using pulsed
355 nm laser excitation with synchronized detection. The novel synchronized detection system allows high signal-tonoise
ratio to be achieved in the resulting autofluorescence signal, which may in turn produce high contrast images that
improve diagnosis, even in the presence of ambient room light. The synchronized set-up utilizes a compact, diode
pumped, pulsed UV laser at 355 nm which is coupled to a CCD camera and a liquid crystal tunable filter. The excitation
and image capture is sampled at 5 kHz and the resulting autofluorescence is captured with the liquid crystal filter cycling
through seven wavelengths between 420 nm and 580 nm. The clinical study targets pigmented skin lesions and evaluates
the prospects of using autofluorescence as a possible means in differentiating malignant and benign skin tumors. Up to
now, sixteen patients have participated in the clinical study. The autofluorescence images, averaged over the exposure
time of one second, will be presented along with histopathological results. Initial survey of the images show good
contrast and diagnostic results show promising agreement based on the histopathological results.
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