The excitation and emission properties of several psoralen derivatives are compared using conventional single-photon excitation and simultaneous two-photon excitation (TPE). Two-photon excitation is effected using the output of a mode-locked titanium: sapphire laser, the near infrared output of which is used to promote nonresonant TPE directly. Specifically, the excitation spectra and excited-state properties of 8-methoxypsoralen and 4'-aminomethyl-4,5,8-trimethylpsoralen are shown to be equivalent using both modes of excitation. Further, in vitro feasibility of two-photon photodynamic therapy (PDT) is demonstrated using Salmonella typhimurium. Two-photon excitation may be beneficial in the practice of PDT because it would allow replacement of visible or UV excitation light with highly penetrating, nondamaging near infrared light and could provide a means for improving localization of therapy. Comparison of possible laser excitation sources for PDT reveals the titanium: sapphire laser to be exceptionally well suited for nonlinear excitation of PDT agents in biological systems due to its extremely short pulse width and high repetition rate that together provide efficient PDT activation and greatly reduced potential for biological damage.

Passively mode-locked titanium:sapphire (Ti:S) lasers are capable of generating a high-frequency train of transform-limited subpico-second pulses, producing peak powers near 105 W at moderate average powers. The low energy per pulse (<20 nJ) permits low fluence levels to be maintained in tightly focused beams, reducing the possibility of saturating fluorescence transitions. These properties, combined with a wavelength tunability from approximately 700 nm to 1 μm, provide excellent opportunities for studying simultaneous two-photon excitation (TPE). However, pulse formation is very sensitive to a variety of intracavity parameters, including group velocity dispersion compensation, which leads to wavelength-dependent pulse profiles as the wavelength is scanned. This wavelength dependence can seriously distort band shapes and apparent peak heights during collection of two-photon spectral data. Since two-photon excited fluorescence is proportional to the product of the peak and average powers, it is not possible to obtain source-independent spectra by using average power correction schemes alone. Continuous-wave, single-mode lasers can be used to generate source-independent two-photon data, but these sources are four to five orders of magnitude less efficient than the mode-locked Ti:S laser and are not practical for general two-photon measurements. Hence, a continuous-wave, single-mode Ti:S laser has been used to collect a source-independent excitation spectrum for the laser dye Coumarin 480. This spectrum may be used to correct data collected with multimode sources; this possibility is demonstrated by using a simple ratiometric method to collect accurate TPE spectra with the mode-locked Ti:S laser. An approximate value of the two-photon cross section for Coumarin 480 is also given.

We demonstrate the use of microcantilevers coated with ultraviolet cross-linking polymers as optical radiation dosimeters. Upon exposure to radiation, a treated cantilever bends due to stress and its resonance frequency increases due to stiffening. These phenomena can be used to develop sensitive radiation dosimeters which respond to radiation affecting the mechanical properties of the selected coating.

The passively mode-locked titanium: sapphire laser provides new opportunities for acquiring two-photon spectral data in the near-infrared, a region not commonly accessible to synchronously pumped dye lasers. This source generates pulses with peak powers near 100 kW at average powers over 1 W and is capable of yielding two-photon signals roughly two orders of magnitude larger than is possible with synchronously pumped dye lasers. However, the multimode output of this laser exhibits significant temporal and spectral pulse profile variations as the laser wavelength is tuned. As a consequence, peak powers of the titanium:sapphire laser can vary independently from average power across the tuning range. This wavelength dependence, coupled with the quadratic dependence of the two-photon signal upon the instantaneous power of the laser, precludes simple average power correction of nonlinear spectral band shapes. Here, we investigate the key properties of the titanium:sapphire laser as an excitation source for two-photon spectroscopy. We also identify a chemical reference suitable for obtaining source-corrected excitation spectra in the near-infrared using a double-beam, ratiometric approach; this is based on a source-in-dependent two-photon excitation spectrum for the laser dye coumarin-480 that has been obtained with a single-frequency titanium:sapphire laser. From these data, correction factors are generated for correction of multimode source data.

Treatment of cutaneous melanoma (M-3 and B16-F10 implanted in mice) with rapidly-scanned, tightly-focused near infrared light elicits selective destruction of tumor tissue. A single laser treatment yielded complete eradication in >90% of B16-F10 tumors with thicknesses of approximately 3 mm; amelanotic M-3 tumors proved less responsive (ca 25% clearance rate). In addition to local tumor destruction, laser treatment of B16-F10 tumors in immunocompetent mice stimulated enhanced cytokine levels (interleukin-2 and interleukin-10) within treated tumor tissues and rejection of tumor cells upon a subsequent challenge dose. Such an antitumor immune response may lead to improved outcomes at both the treatment site and at sites of distant metastasis.

Multiphoton microscopy is a powerful tool for imaging sub-cellular distribution ofluminescent compounds present in living cells. We have used this tool to study the distribution and pharmacology of photosensitizers in tissue and tissue culture. Murine hepatoma tumor cells dosed with a photosensitizer were briefly photoactivated, then imaged for periods up to several hours. Using the photosensitizer Rose Bengal with green light activation, nearly immediate photolytic release of lysosomal enzymes resulted in catastrophic cell destruction within 5-30 minutes. The magnitude and rapidity of this response is markedly different than that observed with other photosensitizer agents, and is consistent with in vivo studies illustrating that Rose Bengal is capable of causing extremely rapid destruction of treated tumors.

Spatlal fllterlng of two-photon exclted fluorescence comblned wlth wcond harmonlc detection has duinonstrated a redduc#on ln the rlgnal blank resuHlng In Improved detectkn Iknlts. The second harmonlc detectkn fluorometer k basad on the recovery of the second harmonlc produced as a resun of nonhar abrorptbn of two photons from a dnwddaUy moddated laser. Thk Instrument k capable of acqulrlng corrected twoghoton exdtatlon spectra rapidly w h h shnultaneousty provkllng detectlon llmltr as low as 200 pM (S/N = 3)fwstwmgakorknruch as^^^ wlth an 82 MHr traln of 12-p u h havlng an average power of 180 mW. Varlour Instrumental contrlbutlons to the ndw spectrum are examined. A tltanlum:sapphke laser was usod io demonstrate the compatlblllty of very rhort laser pulses wlth second harmonic detectlon. A detectbn llmn of 7.55 nM was 0btaiCl.d for the l a m dye coumarln-480 (WN = 9) wlth an 82 MHz traln of 1 -p u h havlng an average power of 850 mW.

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