We compare the emission spectra and analytical response profiles of several semiselective luminescent probes that are commonly used to quantify nonfluorescent analytes (ruthenium(II) tris(2,2′-bipyridyl) dication, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dication, SNARF<sup>®</sup>-1, fluorescein, Rhodol Green™, and Sodium™, Magnesium™, and Calcium Green™) when they are excited under one- and two-photon excitation conditions in water, aqueous solutions of dyes or fluorophores, undiluted mouse or human serum, human urine, and mouse whole blood. The results from this work can be summarized as follows. First, in cases where the probes possess ground states that are composed of multiple species in equilibrium (e.g., acid and base forms of a luminophore), the analyte-dependent emission spectra can be significantly different under one- and two-photon excitation conditions due to differences in the relative one- and two-photon cross-sections associated with the individual ground-state species. Second, for those probes that exhibit one- and two-photon dependent emission spectra, an assessment of the analytical response vs. target analyte concentration profiles show that the response dynamic range can be improved by up to 100 fold, but the response sensitivity can decrease by up to 25% for two- vs. one-photon excitation. Third, two-photon excited luminescence provides a means to use these semiselective luminescent probes to quantify nonfluorescent analytes directly within optically dense mixtures, including solutions of dyes, mouse or human serum, human urine, and mouse whole blood. Finally, two-photon excited luminescence allows one to quantify nonfluorescent analytes in a spatially defined manner within complex samples.

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