The influence of intracellular mTHPC concentration upon photobleaching dynamics
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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