Two-Color Two-Photon Excitation Using Femtosecond Laser Pulses Stefan Quentmeier, ² Stefan Denicke, ² Jan-Eric Ehlers, ² Raluca A. Niesner, ‡ and Karl-Heinz Gericke* ,² UniVersity of Braunschweig, Institute for Physical und Theoretical Chemistry, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany, and Helmholz Zentrum fu ¨r Infektionsforschung, Inhoffenstrasse 7, 38124 Braunschweig, Germany ReceiVed: December 3, 2007; In Final Form: January 30, 2008 The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process. Introduction In recent years, two-photon laser scanning microscopy (2pm) 1 has become an indispensable tool especially for biological science as the use of more than one photon provides numerous advantages. First, separation of excitation and fluorescence light is much easier because of the larger difference in wavelength compared to conventional confocal microscopy. Therefore better signal-to-noise ratios and high-contrast pictures can be obtained. Second, the usage of IR excitation light instead of UV/vis light leads to reduced photo damage and higher penetration depth 2 in biological tissue due to lower absorption coefficients in the IR region 3 than in the visible which is usually used for confocal microscopy. Additionally, due to the quadratic dependence on the intensity, excitation in 2pm is limited to the focal area only. Thus, out-of-focus excitation is avoided leading to limited photo damage and an intrinsic 3D resolution. Most common excitation sources for 2pm are Ti:sapphire (Ti:Sa) lasers. The femtosecond (fs) pulses from these lasers are almost ideal for this purpose as they combine high excitation power during the pulse, which is essential for a good excitation yield, 4 with low-energy deposition to the sample preventing it from being photo damaged. Furthermore, pulsed excitation furnishes the basis for excellent time resolution using time-gated 5 or time-correlated single-photon-counting (TCSPC) 6 detection systems. Hence, a variety of time-resolved fluorescence techniques such as fluo- rescence lifetime imaging (FLIM) 5,7-9 or fluorescence anisot- ropy imaging (Tr-FAIM) 8 have been established. Typically, 2pm and other spectroscopic two-photon methods are performed almost exclusively using two excitation photons of the same wavelength. Only very few attempts have been made so far to extend these methods to two-color two-photon excitation. 10-12 This is surprising as the use of two photons at different wavelengths for excitation offers the opportunity to extend the wavelength range where the Ti:Sa laser can be used under retention of all advantages of two-photon excitation. According to theoretical considerations, spatial resolution 13 and penetration ability, 14 depending on the microscopical setup, are expected to improve for 2c2p excitation compared to confocal or conventional 2pm microscopy, respectively. Using the fundamental and second harmonic of a Ti:Sa laser, 2c2p excitation in the UV can be achieved. As modern mode- locked femtosecond lasers offer a tunable range from about 700 to 1000 nm, the conventional 1c2p technique excites fluoro- phores in a spectral range of about 350-500 nm. With 2c2p, an additional spectral window from about 230 to 330 nm is at hand extending the wavelength range of the Ti:Sa laser to the UV where, for example, tryptophan, a fluorescent amino acid responsible for protein fluorescence, 15,16 can easily be excited. Furthermore, this spectral window corresponds to the excitation wavelength of DNA. 17 In addition, 2c2p excitation offers a unique opportunity for getting a deeper understanding of two-photon absorption processes. Here, in contrast to 1c2p experiments, it is possible to manipulate the polarization of each of the absorbed photons independently. Theoretical considerations 9,18 as well as 2c2p experiments conducted as absorption measurements 19 imply that there is a close relationship between the relative polarizations of the two photons and the symmetry of the involved electronic states. The absorption data published by McClain 18,19 lead to the assumption that it should be possible to discriminate chromophores possessing different symmetries by their different behavior toward different excitation polarizations. An additional * Corresponding author. E-mail: k.Gericke@tu-bs.de. ² University of Braunschweig, Institute for Physical und Theoretical Chemistry. ‡ Helmholzzentrum fu ¨r Infektionsforschung. 5768 J. Phys. Chem. B 2008, 112, 5768-5773 10.1021/jp7113994 CCC: $40.75 © 2008 American Chemical Society Published on Web 04/12/2008