Porphyrin Bleaching and PDT-induced Spectral Changes are Irradiance Dependent in ALA-sensitized Normal Rat Skin In Vivo¶

Abstract

Photobleaching kinetics of aminolevulinic acid-induced protoporphyrin IX (PpIX) were measured in the normal skin of rats in vivo using a technique in which fluorescence spectra were corrected for the effects of tissue optical properties in the emission spectral window through division by reflectance spectra acquired in the same geometry and wavelength interval and for changes in excitation wavelength optical properties using diffuse reflectance measured at the excitation wavelength. Loss of PpIX fluorescence was monitored during photodynamic therapy (PDT) performed using 514 nm irradiation. Bleaching in response to irradiances of 1, 5 and 100 mW cm-2 was evaluated. The results demonstrate an irradiance dependence to the rate of photobleaching vs irradiation fluence, with the lowest irradiance leading to the most efficient loss of fluorescence. The kinetics for the accumulation of the primary fluorescent photoproduct of PpIX also exhibit an irradiance dependence, with greater peak accumulation at higher irradiance. These findings are consistent with a predominantly oxygen-dependent photobleaching reaction mechanism in vivo, and they provide spectroscopic evidence that PDT delivered at low irradiance deposits greater photodynamic dose for a given irradiation fluence. We also observed an irradiance dependence to the appearance of a fluorescence emission peak near 620 nm, consistent with accumulation of uroporphyrin/coproporphyrin in response to mitochondrial damage.

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

“…Anidjaret al 24 used the ratio of tissue fluorescence at two wavelengths for differentiating neoplastic lesions and normal bladder tissue. In addition, a number of groups [25][26][27][28] reported using the ratio of fluorescence and diffuse reflectance or the variants of the ratio to correct for the effect of tissue absorption and scattering and obtain intrinsic fluorescence ͑see Ref. 29 for a more comprehensive review about this technique͒.…”

Portable instrument that integrates irradiation with fluorescence and reflectance spectroscopies during clinical photodynamic therapy of cutaneous disease

“…The dynamics of this photoproduct include a slow photobleaching during PDT, suggesting a possible role as a sensitizer. It is also notable that the basis set used in the SVD fitting of the BCC data was derived from measurements made on the skin of ALAsensitized rats, 24 and as seen in Fig. 4, these basis spectra provide excellent fits to the measured spectra from human skin.…”

“…The tissue autofluorescence spectrum used in the fitting is the normalized average of 18 spectra excited by 633 nm light measured from the forearms of two volunteers, and the fiber autofluorescence spectrum is the normalized average of 18 spectra measured by directing 633 nm light through the treatment channel of the instrument. The PpIX and photoproduct I basis spectra were obtained in previous work by Finlay et al 24 A 61-term Fourier series is also included in the spectral basis library to identify unaccounted-for contributions in the measured spectra and any possible autofluorescence changes induced by the therapy. A penalizing weight is given to this series, however, so that fitting preference is given to known bases.…”

“…12,22 Other spectroscopically accessible parameters such as the production of photosensitizer photoproducts and the accumulation of endogenous biological fluorophores may offer promise as dose metrics. 23,24 Although fluorescence and reflectance spectroscopy have been used in clinical settings during PDT, we are aware of no device that combines both of these spectroscopies with the delivery of the PDT treatment laser in a single, compact system. In this work, we describe such an integrated system that delivers PDT while utilizing fluorescence spectroscopy to report PpIX and photoproduct contributions to tissue fluorescence and reflectance spectroscopy to measure tissue optical properties.…”

Parameter determination for BPD mediated vascularPDT

“…The spectra were analyzed with a singular value decomposition (SVD) fitting method to separate out autofluorescence and photosensitizer [7]. The in vivo concentration of photosensitizer as obtained by comparing the in vivo spectrum with those measured in phantoms of known photosensitizer concentrations.…”