Detector nonlinearity in frequency-domain fluorometry

Frequency-domain fluorometry relies on the measurement of the phase and amplitudes of the Fourier components of the time-dependent fluorescence signal. Experimental results that show that a conventional photomultiplier is subject to intensity-dependent phase shifts are presented. The measurements indicate that this is a problem well below the maximum linear current of the photomultiplier response. These results have important implications in frequency-domain fluorescence anisotropy experiments, in which the parallel and the perpendicular components of the emission intensity are inherently different from one another: a phase shift can be introduced by the photomultiplier.