We have constructed a new two-photon phase-resolved fluorimeter that can be used to selectively record analyte emission spectra from optically challenging samples (soft drinks, juices, and urine) without sample pretreatment. We demonstrate the use of two-photon-excited phase-resolved fluorescence (TPEPRF) by minimizing hyper-Rayleigh scatter and autofluorescence in real samples. When compared to steady-state two-photon-excited fluorescence, TPEPRF offers an improvement in signal-to-background of one to two orders of magnitude.

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