Photochemistryand Photobiology; 1997, 65(1): 91-95 zyxwvu Research Note Two-Photon Excitation of 4’-Hydroxymethyl-4,5’,8-Trimethylpsoralen Dennis H. Oh*’, Robert J. Stanley2, Michelle Lin’, Warren K. Hoefflerl, Steven G. Boxer2, Michael W. Bern@ and Eugene A. Bauerl ’Department of Dermatology, Stanford University Medical Center Stanford, CA, USA; 2Department of Chemistry, Stanford University, Stanford, CA, USA and 3Beckman Laser Institute, University of California at Irvine, Irvine, CA, USA Received 25 June 1996; accepted 1 October 1996 ABSTRACT zyxwvutsr Psoralens are a class of pharmaceutical agents commonly used to treat several cutaneous disorders. When irradi- ated with a mode-locked titanium : sapphire (Ti : sap- phire) laser tuned to 730 zyxwvuts nm, an aqueous solution of 4‘- hydroxymethyl-4,5’,8-trimethylpsoralen (HMT) emits blue light. The emission spectrum is centered at 452 nm and is identical to that obtained by one-photon excitation with UVA excitation, and its magnitude depends quad- ratically on the intensity of laser excitation. These results suggest that two-photon excitation occurs to a potentially photochemically active state. To estimate the two-photon absorption cross section, it was first necessary to measure the emission quantum yield of HMT using 365 nm exci- tation at room temperature that resulted in a value of zyxwv 0.045 * 0.007. The two-photon absorption cross section of HMT at 730 nm is therefore estimated to be 20 X cm4 s (20 Goppert-Mayer). The excited-state photophys- ics and photochemistry of psoralens suggest potential ap- plications to cutaneous phototherapy in diseases such as psoriasis and dystrophic epidermolysis bullosa. INTRODUCTION zyxwvutsr Psoralens, also known as furocoumarins, are now commonly used in conjunction with 320-400 nm UVAt light to treat a variety of dermatologic disorders such as psoriasis, sclero- derma, cutaneous T-cell lymphoma and vitiligo (1). Psora- lens associate with double-stranded nucleic acids and, upon irradiation with UVA light, will form interstrand crosslinks with thymidine and other pyrimidine bases, thus disrupting *To whom correspondence should be addressed at: Department of Dermatology, Stanford University Medical Center, 900 Blake Wilbur Drive, Rm. W0069, Stanford, CA 94305-5334, USA. Fax: 41 5-123-1796; e-mail: ohd@hosp.stanford.edu. zyxwvutsrq TAbbreviotions: CW, continuous wave; G-M, Goppert-Mayer; HMT, 4’-hydroxymethyl-4,5‘,8-trimethylpsoralen; Rh, rhodamine B; Ti : sapphire, titanium : sapphire; TMP, 4,5’8-trimethylpsoralen; UVA, 320-400 nm radiation. zyxwvutsrqp 0 1997 American Society for Photobiology 003 1-8655/97 $5.00+0.00 normal replication, transcription and recombination (24). Recently, we have shown that it is possible to irradiate psor- alen covalently linked to antisense oligonucleotides to inhibit specifically expression of collagenase from cultured human dermal fibroblasts (5). These data suggest a potential use in gene therapy for diseases such as recessive dystrophic epi- dermolysis bullosa in which collagenase is overexpressed. While the action spectrum of psoralen photochemotherapy has conventionally been regarded as 320-380 nm, recent in- vestigation has determined that 320-335 nm may be more effective (1,6). Ultraviolet A light penetrates farther into the skin because it is scattered and absorbed less than shorter wavelength W B light (290-320 nm), and thus UVA may potentially activate psoralens in several layers of tissue, in- cluding both the intended target and nontarget cells. Addi- tionally, UVA has biological effects independent of psora- len, such as enhanced collagenase expression in fibroblasts and DNA mutagenesis (7.8). Therefore, a form of therapy in which the specific depth of UVA penetration and psoralen photochemistry could be controlled would be useful and generally applicable to phototherapy . Two-photon excitation has recently been explored as a method of selectively irradiating a particular volume of sam- ple, with applications in three-dimensional microscopic im- aging and optical information storage (9,lO). The probability that a molecule absorbs two photons simultaneously depends linearly on its intrinsic two-photon cross section, 6, and quadratically on the incident light intensity (9-1 1). Although the two-photon cross sections of molecules are typically small compared with those for one-photon absorption, two- photon processes become increasingly important at the high light intensities attainable with laser irradiation. In particular, crossed or focused laser beams make two-photon excitation potentially attractive for depth-specific irradiation of photo- active therapeutic agents in tissues such as skin where the cellular targets may occur in discrete layers. Because longer wavelengths of light in the near-infrared portion of the elec- tromagnetic spectrum can be used, two-photon irradiation may potentially avoid the damaging and toxic effects of UVA light if these processes have different two-photon ac- tion spectra. Moreover, the greater depth of cutaneous pen- etration achievable with near-infrared light compared with 91