William J. Cottrell

Irradiance-Dependent Photobleaching and Pain in δ-Aminolevulinic Acid-Photodynamic Therapy of Superficial Basal Cell Carcinomas

Purpose: In superficial basal cell carcinomas treated with photodynamic therapy with topical δ-aminolevulinic acid, we examined effects of light irradiance on photodynamic efficiency and pain. The rate of singlet-oxygen production depends on the product of irradiance and photosensitizer and oxygen concentrations. High irradiance and/or photosensitizer levels cause inefficient treatment from oxygen depletion in preclinical models. Experimental Design: Self-sensitized photobleaching of protoporphyrin IX (PpIX) fluorescence was used as a surrogate metric for photodynamic dose. We developed instrumentation measuring fluorescence and reflectance from lesions and margins during treatment at 633 nm with various irradiances. When PpIX was 90% bleached, irradiance was increased to 150 mW/cm2 until 200 J/cm2 were delivered. Pain was monitored. Results: In 33 superficial basal cell carcinomas in 26 patients, photobleaching efficiency decreased with increasing irradiance above 20 mW/cm2, consistent with oxygen depletion. Fluences bleaching PpIX fluorescence 80% (D80) were 5.7 ± 1.6, 4.5 ± 0.3, 7.5 ± 0.8, 7.4 ± 0.3, 12.4 ± 0.3, and 28.7 ± 7.1 J/cm2, respectively, at 10, 20, 40, 50, 60 and 150 mW/cm2. At 20-150 mW/cm2, D80 doses required 2.5-3.5 min; times for the total 200 J/cm2 were 22.2-25.3 min. No significant pain occurred up to 50 mW/cm2; pain was not significant when irradiance then increased. Clinical responses were comparable to continuous 150 mW/cm2 treatment. Conclusions: Photodynamic therapy with topical δ-aminolevulinic acid using ∼40 mW/cm2 at 633 nm is photodynamically efficient with minimum pain. Once PpIX is largely photobleached, higher irradiances allow efficient, rapid delivery of additional light. Optimal fluence at a single low irradiance is yet to be determined.

Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes

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.

Simulations of Measured Photobleaching Kinetics in Human Basal Cell Carcinomas Suggest Blood Flow Reductions During ALA-PDT

In a recently completed pilot clinical study at Roswell Park Cancer Institute, patients with superficial basal cell carcinoma (sBCC) received topical application of 20% 5‐aminolevulinic acid (ALA) and were irradiated with 633 nm light at 10–150 mW cm−2. Protoporphyrin IX (PpIX) photobleaching in the lesion and the adjacent perilesion normal margin was monitored by fluorescence spectroscopy. In most cases, the rate of bleaching slowed as treatment progressed, leaving a fraction of the PpIX unbleached despite sustained irradiation. To account for this feature, we hypothesized a decrease in blood flow during ALA‐photodynamic therapy (PDT) that reduced the rate of oxygen transported to the tissue and therefore attenuated the photobleaching process. We have performed a detailed analysis of this hypothesis.

Microscope enabling multimodality imaging, angle-resolved scattering, and scattering spectroscopy.

We present the design, construction, and initial characterization of a multifunctional imaging/scattering spectroscopy system built around a commercial inverted microscope platform. The system enables co-registered brightfield, Fourier-filtered darkfield, and fluorescence imaging; monochromatic angle-resolved scattering measurements; and white-light wavelength-resolved scattering spectroscopy from the same field of view. A fiber-based illumination system provides illumination-wavelength flexibility and a good approximation to a point source. The performance of the system in its various data acquisition modes is experimentally verified using fluorescent microspheres. This multifunctional instrument provides a platform for studies on adherent cells from which the biophysical implications of subcellular light scattering can be studied in conjunction with sensitive fluorescence-based techniques.

System for providing simultaneous PDT delivery and dual spectroscopic monitoring in clinical basal cell carcinoma therapy

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.