PpIX induced by administration of ALA is being successfully employed for tissue diagnosis and photodynamic therapy (PDT) of, for example, brain malignancies. To guide tissue biopsy by fluorescence during stereotaxy, correct quantification of the PpIX accumulation is required. However, the detected fluorescence intensity and spectral shape are influenced and distorted by the varying optical properties of tissue. Quantitative PpIX measurements thus need to disentangle these effects in order to provide the undistorted, intrinsic fluorescence. Numerous methods for obtaining the intrinsic fluorescence have been developed and optimized for certain fluorochromes. PpIX poses a particular case where excitation and fluorescence are spectrally well separated. Furthermore, the fluorescence appears within the red wavelength region where absorption in tissue is relatively weak.
Here, three experimental approaches towards assessing the intrinsic fluorescence for PpIX in homogeneous phantom materials for four subsets at tissue-like conditions, were tested and compared; 1) single-fiber with diameters 200-800 μm, 2) a two-fiber probe with evaluation based on an empirical ratio between fluorescence and reflectance signals or 3) a multi-fiber probe for combined fluorescence and reflectance measurements with evaluation based on a theoretical model of light propagation. All methods could be realized with an outer probe diameter of less than 1.5 mm, thus applicable during stereotaxy. Method 3 could quantify the PpIX concentration best, regarding all four subsets and thus covering a broad, physiologically relevant range of optical parameters. With accuracies between ± 3.2 % and ± 24.8 % for different subsets it was overall a great improvement to the accuracies resulting from calculations based on a plane wave geometry, which vary from ± 4.6 % to ± 84.3 %.