A fluorescence instrument response is the distribution of recorded delays for an effectively prompt optical signal. Finite excitation-pulse width and timing variation in the photodetector and electronics contribute. With a normalized response , intrinsic fluorescent photon rate , and background rate , the expected trace is
(1)
Fit a candidate decay after this convolution with the measured response; treating the broadened leading edge as the intrinsic decay can bias fluorescence lifetime. The accessible technical note describes measuring prompt scattered excitation with a nonfluorescent scattering sample.[ref]
Figure 1.
Synthetic fluorescence lifetime decay broadened by an instrument response
. Original stipulated model, not measurements: for and zero otherwise; is a normalized Gaussian function of standard deviation centred at zero; the background is in the common signal units. The left panel compares intrinsic and convolved traces; the right shows how an unremoved background flattens the tail on a logarithmic scale. Negative delays come from the stipulated timing response about the pulse reference, not emission before excitation. Only the instrument-response curve is rescaled to unit peak for display. Details.

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  1. Time-correlated single-photon counting
  2. Fluorescence lifetime
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