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Metal ion-responsive fluorescent probes are powerful tools for visualizing labile metal ion pools in live cells. To take full advantage of the benefits offered by two-photon excitation microscopy, including increased depth penetration, reduced phototoxicity, and intrinsic 3D capabilities, the photophysical properties of the probes must be optimized for nonlinear excitation. This review summarizes the challenges associated with the design of two-photon excitable fluorescent probes and labels and offers an overview on recent efforts in developing selective and sensitive reagents for the detection of metal ions in biological systems.

Two novel donor-acceptor-substituted tetrahydropyrene derivatives (1 and 2, Chart 1) were synthesized and photophysical properties investigated in solvents of different polarities. These studies revealed the existence of an intramolecular charge transfer (ICT) excited state in these molecules. The fluorescence lifetime and quantum yield measurements revealed that emission occurs from a planar 1 CT state in all the solvents. The solvent-dependent Stokes shift values for 1 and 2 were analyzed by the Lippert-Mataga equation and Liptay's modified equation to obtain the excited-state dipole moment values. The CT nature of the emitting states is further confirmed by studies in acidic medium. Change of pH results in well-separated LE and CT fluorescence and absorption bands. It is suggested that this property can be utilized for pH-sensing applications. † This is publication number PPD-(RRLT)-162 from the Photosciences and Photonics Division, Regional Research Laboratory (CSIR) Trivandrum.

Two-photon excitation fluorescence microscopy (TPEM) has rapidly evolved into a widely used tool in biological and biomedical research. 1 Compared to traditional fluorescence microscopy, TPEM offers intrinsic 3D resolution combined with reduced phototoxicity, increased specimen penetration, and negligible background fluorescence. At present, most fluorophores used as labels or sensor platforms in TPEM have been adopted from linear microscopy and are not optimized for two-photon excitation. 2 Notably, the fluorescence brightness (ηδ), defined by the product of TPA cross section (δ) and emission quantum yield (η), is typically low due to a modest δ. 3 The development of new TPEM-optimized fluorophores is particularly vital in the context of biological metal-ion sensing since most of currently available ratiometric sensors, including the widely used dyes fura-2 and indo-1, 4 exhibit a low brightness that decreases even further upon cation binding. In addition, the majority of ratiometric metal-ion sensors offer only a large shift of the excitation peak but not emission energy. If only a single two-photon excitation source is on hand, such sensors are not suitable for dynamic ratiometric TPEM imaging with temporal resolution. In this communication, we address these problems with a molecular design approach that yields both an increase in δ and a shift of the peak emission energy upon metal-ion binding in a polar environment.Molecular design strategies of organic molecules with large δ are well-established. 5,6 In general, the magnitude of δ increases with an increasing degree of intramolecular charge transfer (ICT) upon excitation. For example, centrosymmetric fluorophore architectures consisting of a conjugated linear π-system with an acceptor moiety sandwiched between two electron donors (D-π-A-π-D) exhibit exceptionally large δ values. 6 However, in water, the highly polarized excited state gives rise to enhanced solvent-solute interactions, which in turn leads to a reduced δ, more efficient nonradiative deactivation, and thus to a drastically reduced brightness. 7 The centrosymmetric architecture poses additional challenges for the design of metal-ion sensors: (1) the interpretation of the sensor response is complicated due to the presence of two metal binding sites (Scheme 1a); (2) metal binding induces a reduction of ICT which in turn results in a smaller δ accompanied by a strongly blue-shifted peak excitation energy; and (3) partial decomplexation or ejection of the metal cation in the excited state is presumably responsible for typically small emission shifts that are not suitable for ratiometric sensing. 8 Here, we propose to overcome these problems by using a simplified D-A 9 motif where the metal-ion binds to the acceptor rather than donor site (Scheme 1b). Besides eliminating the second binding site, such an arrangement should yield an increase rather than decrease of ICT upon excitation. As a consequence, metal-ion binding is expected to result in an increased δ, enhanced fluorescence brightness, and a ...