The linear dependence of TPE probability on the TPE cross section 
(see Equation 1) underscores the importance of knowing the TPE cross sections and spectra for biologically useful fluorophores. Knowing the absolute values of TPE cross sections and their wavelength dependence is useful to design a particular experiment. For example, use of a calcium-sensitive fluorophore with a relatively high TPE cross section will facilitate the study of intracellular calcium dynamics deep in biological specimens compared with a fluorescent calcium indicator with lower two-photon absorption; and in studies using two-photon fluorescence resonance energy transfer (FRET), the wavelength exciting the donor molecule may not excite the acceptor molecule.32 Methods to determine the TPE action cross section are technically demanding and are described in detail elsewhere.16,33,34 TPE spectra for a number of fluorophores in the spectral range of 
700 to 1050 nm have been published.34,35 TPE cross section values at the peak absorption wavelength vary from 10–2 GM for NAD(P)H35 to 50 000 GM for cadmium selenide-zinc sulfide quantum dots.33 The values for most of the commonly used fluorophores, including those of conventional calcium indicators, are in the range of 1 to 300 GM.16,35 TPE action cross sections and their wavelength dependence may differ in vitro and in vivo, because of protein binding28 or pH changes.36 Green fluorescent protein (GFP) and its blue- and red-shifted variants have relatively large TPE action cross sections in the range of 100 to 200 GM, making these proteins well suited for two-photon microscopy in living specimens.2,3,37 Although large TPE cross section values are desirable for TPE fluorescence microscopy, the probability of excitation saturation increases with increasing action cross section, resulting in an increase in TPE volume and, consequently, a decrease in optical resolution with increasing illumination intensity.12,33
No differences between one- and two-photon–excited fluorescence emission spectra have been found so far,16 and there is usually substantial overlap of the two-photon-action cross section and the single-photon excitation spectrum when plotted at twice the wavelength. However, large blue shifts have been observed for rhodamine and several ion-sensitive fluorophores. As one consequence, UV-light excitable indicators such as fura-2 (and other members of the fura family)34 and indo-138 for Ca2+ and SBFI for Na+29 have become accessible for two-photon fluorescence microscopy. As another consequence, a number of fluorophores with disparate one-photon excitation spectra can be excited simultaneously by TPE at a single wavelength, thereby avoiding chromatic aberration (Figure 3). Combined with the large separation between the excitation and emission light, this feature facilitates multicolor fluorescence imaging, provided that the emission spectra of the dyes used are well separated. Figure 3 shows simultaneous TPE of the calcium indicator rhod-2 and enhanced GFP (EGFP). When simultaneously exciting multiple fluorophores, the unwanted existence of FRET between them should be examined carefully.
Although TPE at wavelengths twice the single-photon absorption wavelength in general results in fluorescence light sufficient for TPE laser scanning microscopy, evaluation of the TPE spectrum of a dye used in a particular experiment may be helpful to further enhance brightness for imaging. For example, Kuhn et el recently found that TPE of the novel voltage-sensitive dye ANNINE-6 at wavelengths more than twice the published single-photon excitation wavelengths increased voltage-sensitivity of fluorescence changes by a factor of 2 without evidence for photobleaching.39 To exploit the full potential of TPE microscopy, it would be desirable to synthesize fluorescent molecules with large TPE cross sections, similar to those of C625 (744 GM)40 and water-soluble quantum dots (50 000 GM).33 Excitation and emission spectra of fluorescent molecules can be insufficiently distinct to be concurrently imaged by conventional means. Recent advances in imaging technology that exploit spectral and fluorescence lifetime fingerprinting to separate closely overlapping fluorescent indicators, such as linear unmixing,41–43 allow signals from different fluorescent markers to be resolved. This approach offers the opportunity to simultaneously image multiple colored, spectrally overlapping markers within living cells and tissue.
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