Minimizing urine autofluorescence under multi-photon excitation conditions

We report on the effects of excitation wavelength, laser power, and phase resolution on the multi-photon-excited autofluorescence (background) from human urine. When compared to the autofluorescence under one-photon excitation conditions (lambda(ex) = 260-480 nm), the urine multi-photon-excited autofluorescence (lambda(ex) = 725-950 nm) can be less complicated. However, at higher laser powers, the multi-photon-excited autofluorescence spectra that are produced by excitation above ~775 nm are more complex in comparison to the corresponding one-photon-excited autofluorescence. The origin of these more complex spectra arises from simultaneous two- and three-photon-driven excitation of intrinsic luminescent species within the urine. At lower laser powers, three-photon-driven processes are minimized and the autofluorescence spectrum is simplified. Phase resolution is used to further minimize the urine autofluorescence, but it cannot fully eliminate autofluorescence even when excitation is performed under multi-photon conditions at 950 nm. For detecting 250 nM Rhodamine 6G (a mock analyte) dissolved in urine, we find that the two-photon excitation is superior in comparison to one-photon excitation by 5- to 70-fold, depending on the excitation wavelength. Phase resolution combined with two-photon excitation leads to an additional 5- to 7-fold improvement in signal-to-background ratios in comparison to steady-state two-photon excitation.