Intracellular Photobleaching of 5,10,15,20-Tetrakis(m-hydroxyphenyl) chlorin (Foscan®) Exhibits a Complex Dependence on Oxygen Level and Fluence Rate¶

Abstract

The understanding of photosensitizer photobleaching is important not only for mechanistic studies, but also for the development of monitoring techniques for clinical dosimetry in photodynamic therapy. In this study, we investigated the intracellular photobleaching of 5,10,15,20‐tetrakis(m‐hydroxyphenyl)chlorin (mTHPC, Foscan®) in the murine macrophage cell line J774A.1, using quantitative fluorescence imaging microscopy, microspectrofluorometry and microspectrophotometry. Using 652 nm laser irradiation, it was found that mTHPC exhibits oxygen‐ and fluence rate–dependent intracellular photobleaching. The kinetics showed an inverse dose–rate behavior, i.e. a reduction of fluence rate resulted in more photobleaching at comparable fluences. The effect of deoxygenation was found to be more complex, with decreased bleaching at low fluence rates and increased bleaching at higher fluence rates. The intracellular formation of reactive oxygen species was measured using 2′,7′‐dichlorodihydrofluorescein diacetate. The results are analyzed in terms of competitive Type‐I and Type‐II mechanisms.

Photodynamic therapy of newly implanted glioma cells in the rat brain

“…Of particular importance is the observation that PDT-induced cytotoxicity is critically dependent on both radiant exposure and the rate of light delivery (irradiance)-lower irradiances are more effective, especially at low radiant exposures. The decreased efficacy of higher irradiances observed in BT 4 C monolayers is in good agreement with the findings of other investigators using a variety of cell lines and photosensitizers [25,26]. Oxygen depletion, which has been shown to be important in fluence rate effects observed in vivo, is likely not involved.…”

Calculation of Singlet Oxygen Dose from Photosensitizer Fluorescence and Photobleaching During mTHPC Photodynamic Therapy of MLL Cells¶

“…Thus, for initially well‐oxygenated samples, large changes in [ 3 O 2 ] do not significantly change the value of Λ oxy , provided that [ 3 O 2 ] ≫ k p /k ot (8.7 μ M for mTHPC). The lack of significant oxygen depletion for high fluence rates or high sensitizer concentrations explains the absence of a fluence rate effect observed in other in vitro investigations of mTHPC PDT (40, 41). For the experiment where the oxygen has been depleted by nitrogen bubbling, the singlet oxygen dose has been overestimated by approximately 400%.…”

Precision of light intensity measurement in biological optical microscopy

“…Hence, the technique may be used with biological specimens, which typically exhibit spatially and temporally heterogeneous fluorescence. Furthermore, the specimens may be imaged under non‐ideal (realistic) conditions which involve photobleaching of fluorescent labels (Song et al , 1995; Van Oostveldt et al , 1998; Kunz & MacRobert, 2002; Bernas et al , 2004) and instability of illumination source (Zucker & Price, 2001b).…”