Assessment of One- and Two-Photon Excited Luminescence for Directly Measuring O2, pH, Na+, Mg2+, or Ca2+ in Optically Dense and Biologically Relevant Samples
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®-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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