The influence of intracellular mTHPC concentration upon photobleaching dynamics

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Summary

In this study, we report the effect of the local photosensitizer concentration upon the dynamics of the singlet oxygen-mediated photobleaching, within formalin-fixed keratinocytes. Although the cells were incubated at a single mTHPC dose, cell-to-cell variations in concentration were defined within the perinuclear region by differences in the initial amplitude of the laser-induced fluorescence emission, located around 652 nm. At a fixed laser fluence-rate, it was found that the photobleaching, when plotted as a function of delivered light dose, proceeded more rapidly at higher drug concentration. The mTHPC spectral emission profile is shown to be approximately Lorentzian and remains unchanged as the photobleaching proceeds. This indicates that there is no perturbation of the detected signal due to the inner-filter effect.

Introduction

It is well established that photobleaching is confined to regions of singlet oxygen abundance [1]. Moreover, photosensitizer degradation results in decreased photodynamic oxygen consumption with important consequences for the spatial distribution of photodynamic dose. In this study we describe the influence of the local, intracellular photosensitizer concentration, upon the dynamics of photobleaching. The experiments were performed using formalin-fixed keratinocytes that had been incubated with the photosensitizer mTHPC. The local concentration of photosensitizer, within the detection volume of the 410 nm laser focus, is assumed to be proportional to the initial magnitude of the recorded fluorescence emission.

Section snippets

Cells and culture conditions

Cells used in this experiment were keratinocytes derived from human foreskin. The culture conditions and Foscan administration are discussed in detail in our previous publication [2]. The formalin-fixing procedure was necessary in order to allow prolonged laser exposure at defined points within each cell.

Illumination set up

As reported previously [2], a commercial Raman spectrometer (System 1000, Renishaw plc, Wotton-under-Edge, UK) has been adapted to perform high sensitivity, fluorescence microscopy, allowing

Continuous exposure at constant irradiance

As illustrated in Fig. 1, there is a progressive depletion of the 652 nm emission peak during continuous irradiation with 410 nm laser light. Fig. 2(a) shows the changes in fluorescence intensity as a function of time, recorded from a set of seven cells (Group 1). There is a significant variation in the magnitude of the fluorescence from different cells (normalised mean = 1 ± S.D., .23 around the mean for the first spectrum in each sequence). The same data when plotted versus dose after normalisation

Discussion

The aim of this study was to analyze the effects of photosensitizer concentration in mTHPC induced photobleaching. Previously, Fuchs and Thiele [3] demonstrated that within single cells there are large oxygen concentration gradients, especially close to the mitochondria. Similarly, large variations in oxygen concentration are also observed over small distances in skin, which possesses a complex morphological structure and angioarchitecture.

On the basis of the observed sensitizer fluorescence

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