Angularly resolved light scattering and wavelength-resolved darkfield scattering spectroscopy measurements were performed on intact, control EMT6 cells and cells stained with high-extinction lysosomal- or mitochondrial-localizing dyes. In the presence of the lysosomal-localizing dye NPe6, we observe changes in the details of light scattering from stained and unstained cells, which have both wavelength- and angular-dependent features. Analysis of measurements performed at several wavelengths reveals a reduced scattering cross section near the absorption maximum of the lysosomal-localizing dye. When identical measurements are made with cells loaded with a similar mitochondrial-localizing dye, HPPH, we find no evidence that staining mitochondria had any effect on the light scattering. Changes in the scattering properties of candidate populations of organelles induced by the addition of an absorber are modeled with Mie theory, and we find that any absorber-induced scattering response is very sensitive to the inherent refractive index of the organelle population. Our measurements and modeling are consistent with EMT6-cell-mitochondria having refractive indices close to those reported in the literature for organelles, approximately 1.4. The reduction in scattering cross section induced by NPe6 constrains the refractive index of lysosomes to be significantly higher. We estimate the refractive index of lysosomes in EMT6 cells to be approximately 1.6.
Photodynamic therapy using 5-aminolevulinic acid is an effective therapy for treating basal cell carcinoma, characterized by high lesion clearance and excellent cosmetic outcomes. Treatment optimization and lesion-tailored treatments making use of real-time treatment assessment promise still greater efficacy and improved comfort for patients. In order to monitor treatment parameters during therapy, instrumentation of our own design delivers a 633 nm treatment beam while simultaneously collecting fluorescence spectra. Fluorescence spectra from 650-800 nm are corrected for the effects of tissue optical properties and report protoporphyrin IX (PpIX) photobleaching as well as photoproduct dynamics in the lesion and in the perilesion margin during therapy. Brief treatment interruptions are made for acquisition of white light reflectance spectra from 420-800 nm that are used to generate corrections to fluorescence spectra and can be used to deduce blood volume and hemoglobin oxygen saturation. LabVIEW and Matlab scripts are used for real-time data analysis. Measurements have been made on 5 patients (7 BCC lesions) with a treatment fluence rate of 150 mW cm-2 and on 5 additional patients (5 BCC lesions) at 10 mW cm-2. Measurements are made for each lesion until greater than 90% photobleaching of PpIX is detected at which point the balance of the prescribed fluence is delivered at 150 mW cm-2 without interruption. PpIX bleaching rates between the two fluence rates varied significantly. These measurements were carried out during ALA-PDT treatment of BCC as part of a pilot study designed to guide treatment fluence and fluence rates in an anticipated clinical trial.