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Fluence-rate effects upon m-THPC photobleaching in a formalin-fixed cell system
M Atif1, M R Stringer2, J E Cruse-Sawyer3
1Department of Physics, University of Hull, Cottingham Road, Hull HU67RX, UK.
Abstract:
We have applied a micro-spectroscopic technique in order to record the laser-induced fluorescence emission of the PDT photosensitiser m-THPC (Foscan) from micron-scale locations within individual formalin-fixed keratinocytes. We demonstrate that m-THPC is highly photolabile in this cellular environment, and that the process of photobleaching can be monitored via the depletion in fluorescence emission during continuous irradiation with 410nm laser light. The progressive reduction of the characteristic 652nm m-THPC fluorescence peak can be described with bi-exponential decay kinetics, consistent with a singlet oxygen-mediated process. The rate of photobleaching, when plotted as a function of light dose, shows inverse fluence-rate dependence. Specifically, the rate of photobleaching induced by the higher laser powers appears to be limited by oxygen availability, as demonstrated by an increase in the (1/e) bleaching dose. Fractionated irradiation provides evidence of intracellular re-oxygenation. These results are in qualitative agreement with previous in vitro and in vivo studies, which indicate that the photodynamic dose delivered during light irradiation is critically dependent upon local fluence rate and oxygen partial pressure.
Insights
Micro-spectroscopy reveals that m-THPC (Foscan) photobleaching in keratinocytes depends on oxygen levels. Fractionated light exposure suggests intracellular re-oxygenation, crucial for photodynamic therapy efficacy.
Area of Science:
- Biophysics
- Photochemistry
- Cell Biology
Background:
- Photodynamic therapy (PDT) uses photosensitizers activated by light to generate cytotoxic species.
- m-THPC (Foscan) is a photosensitizer used in PDT, but its behavior in cellular environments needs further characterization.
- Understanding photosensitizer photobleaching is key to optimizing PDT dose delivery.
Purpose of the Study:
- To investigate the photobleaching kinetics of m-THPC in individual formalin-fixed keratinocytes using micro-spectroscopy.
- To determine the influence of light dose, fluence rate, and oxygen availability on m-THPC photobleaching.
- To explore the effects of fractionated irradiation on photobleaching and potential re-oxygenation.
Main Methods:
- Application of micro-spectroscopic technique to record laser-induced fluorescence emission of m-THPC.
- Irradiation of cells with 410nm laser light to monitor fluorescence depletion.
- Analysis of fluorescence decay kinetics and plotting photobleaching rate against light dose.
- Investigating inverse fluence-rate dependence and effects of fractionated irradiation.
Main Results:
- m-THPC demonstrated high photolability within keratinocytes.
- Photobleaching followed bi-exponential decay kinetics, indicative of singlet oxygen involvement.
- Photobleaching rate showed inverse fluence-rate dependence, suggesting oxygen limitation at higher laser powers.
- Fractionated irradiation indicated intracellular re-oxygenation.
Conclusions:
- m-THPC photobleaching is significantly influenced by local oxygen partial pressure and light fluence rate.
- These findings align with previous studies highlighting the critical role of oxygen and light parameters in PDT.
- Optimizing PDT protocols requires careful consideration of these micro-environmental factors for effective treatment.
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