We present absolute two-photon absorption (2PA) spectra of 15 commercial organic dyes covering an extended range of excitation wavelengths, 550-1600 nm. The 2PA is measured with an estimated accuracy +/-10% using a femtosecond fluorescence excitation method. The data are corrected for the variations of the pulse duration and the beam profile with the excitation wavelength, and are applicable as reference standards for 2PA measurements.

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest, and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right FP and excitation wavelength for two-photon applications.Two-photon laser scanning microscopy (2PLSM) 1,2 of cells and tissues expressing fluorescent proteins is becoming a powerful tool for biological studies at different levels of organization [2][3][4] . The advantages of two-photon excitation (2PE) include reduced out-offocus photobleaching, less autofluorescence, deeper tissue penetration, and intrinsically high three-dimensional resolution 1,2 . 2PLSM should make it possible to obtain even better optical recordings of ion concentration and cell signaling with genetically targeted sensors 5,6 . Twophoton excitation of fluorescent proteins can also be considered as potentially advantageous in the contexts of genetically targeted deep photodynamic therapy or chromophore-assisted light inactivation 6 , three-dimensional optical memory 7 , as well as superresolution (subdiffraction limited) imaging techniques, such as stimulated emission depletion 8 , photoactivated localization microscopy, and stochastic optical reconstruction microscopy 9 .To fully realize the potential of 2PE of the fluorescent proteins, it is important to know their two-photon absorption (2PA) spectra, cross sections, σ 2 , and 2PE action cross sections, or brightness, σ 2 ', (σ 2 ' = σ 2 × φ, where φ is the fluorescence quantum yield). The linear, onephoton absorption (1PA) spectra and extinction coefficients of many fluorescent proteins have been described and reviewed 5,6,10 (Supplementary Table 1 online), but the 1PA properties are not sufficient to predict the key 2PA properties such as optimum excitation wavelength and maximum brightness (Box 2).Correspondence should be addressed to M.D. (drobizhev@physics.montana.edu).. 4 Present address: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia. 5 Present address: Vollum Institute, Oregon Health and Science University, Portland, Oregon. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptHere we present a systematic characterization of the 2PA properties of 48 different fluorescent proteins using the same experimental setup, common 2PA reference standards, and an all-optical method for measuring mature chromophore concentration 11 (Supplementary Methods online). Briefly, we use a relative fluorescence method with femtosecond excitation and coumarin 485 (Exciton), coumarin 540A (Exciton), rhodamine 610 (Exciton), fluorescein (Aldrich), and styryl 9M (Aldrich) as 2PA standards 12 , which eliminates the necessity to calibrate the laser parameters. The power dependence of the fluorescence signal was quadratic for all data ...

The spatial control of optical absorption provided by twophoton excitation (TPE) has led to tremendous advances in microscopy 1 and microfabrication 2 . Medical applications of TPE in photodynamic therapy (PDT) 3,4 have often been suggested 5-18 , but have been made impractical by the low twophoton cross-sections of photosensitiser drugs (i.e. compounds taken up by living tissues that become toxic on absorption of light). The invention of efficient two-photon activated drugs will allow precise manipulation of treatment volumes in three dimensions, to a level unattainable with current techniques. Here we present a new family of PDT drugs designed for efficient TPE, and use one of them to demonstrate selective closure of blood vessels via TPE-PDT in vivo. These conjugated porphyrin dimers have two-photon cross-sections that are more than two orders of magnitude greater than those of clinical photosensitisers 17 . This is the first demonstration of in vivo PDT using a photosensitiser engineered for efficient two-photon excitation.Photodynamic therapy is used to treat diseases characterised by neoplastic growth including various cancers, age-related macular degeneration (AMD) and actinic keratosis 3,4 . Cell death is induced by photoexcitation of a sensitiser, generally via production of singlet oxygen. In the absence of light the photosensitiser is benign, so systemic toxicity is rare and treatment may be repeated without acquired resistance. Two-photon excitation of the photosensitiser should allow greater precision than is attainable by conventional one-photon excitation, as a consequence of the quadratic dependence of TPE on the local light intensity -the amount of TPE is inversely proportional to the fourth power of the distance from the focus. In addition, the longer wavelengths associated with TPE allow treatment deeper into tissue, by minimising absorption from endogenous chromophores.High instantaneous photon densities are essential for two-photon excitation. Early TPE-PDT studies used nanosecond lasers, but the dominant effect was photothermal damage [5][6][7] . The advent of commercial femtosecond tuneable Ti:sapphire lasers has greatly facilitated the investigation of TPE-PDT, and the limiting factor has become the availability of suitable photosensitisers. The majority of chromophores possess low two-photon cross-sections, of the order of 1-100 Goeppert-Mayer units (1 GM = 10 -50 cm 4 s photon -1 ). For example, the two FDA-approved PDT photosensitisers, verteporfin and Photofrin (cross sections 50 GM and 10 GM respectively) 17 , are unlikely to be suitable for TPE-PDT, as the high light intensities needed to achieve a therapeutic effect are close to the thresholds for photothermal or photomechanical damage 18 .Several design strategies for TPE-PDT photosensitisers have been reported recently [11][12][13][14][15][16] , but few of these compounds have yet been studied in vitro 15 , and, to date, none have progressed to in vivo testing. Porphyrin derivatives are often effective PDT agents, as exemplified ...

Two-photon absorption spectra (2PA) of a series of conjugated dimers and the corresponding monomer were studied in the near-IR region. All of the dimers show very large peak cross section values, sigma(2) = (3-10) x 10(3) GM (1 GM = 1 x 10(-50) cm(4) s photon(-1)), which is several hundred times larger than that obtained for the corresponding monomer in the same region. We explain such dramatic cooperative enhancement by a combination of several factors, such as strong enhancement of the lowest one-photon Q-transition, better resonance conditions in the three-level system, dramatic enhancement of the excited-state singlet-singlet transition, and parallel arrangement of consecutive transitions in dimers, as compared to perpendicular arrangement in the monomer. We show that the absolute values of the 2PA cross section in these molecules are quantitatively described by a quantum-mechanical expression, derived for the three-level model. We also demonstrate the possibility of singlet oxygen generation upon one- and two-photon excitation of these dimers, which makes them particularly attractive for photodynamic therapy.