There are two ways to calculate the fluorescence lifetime. The quantum efficiency (Q), absorption coefficient (A) and concentration (C) of your sample will not affect the outcome of either method. This is because these characteristics alter the light intensity, which is not a factor in the lifetime calculations.

Schematic overview of factors that are important for calculating the fluorescence lifetime.

Schematic overview of factors that are important for calculating the fluorescence lifetime.

When calculating the fluorescence lifetime from the phase, only the shift in phase between the excitation and emission light is taken into account. The amplitude of the modulated light intensity does not affect the phase shift.

The fluorescence lifetime calculated from the demodulation (M) is also not influenced by the quantum efficiency, the absorption coefficient or concentration of your sample. These factors determine the light intensity emitted by the sample:

\begin{equation}I_{\text{emission}} = Q\cdot A\cdot C\cdot I_{\text{excitation}}.\end{equation}

A lower quantum efficiency, absorption coefficient or concentration results in a lower intensity of the emitted fluorescence light. This affects the average light intensity of the modulated light as well as the amplitude of its variation by the same amount. The modulation (m) is the ratio of the variation of the light intensity (a and b in the figure above) to its average (c and d in the figure):

\begin{equation} m = \frac{Q\cdot A\cdot C\cdot a}{Q\cdot A\cdot C\cdot c} = \frac{a}{c}. \end{equation}

The quantum efficiency, the absorption coefficient and the concentration affect the variation and the average by the same amount. These factors cancel in the calculation of m and are therefore of no influence to the lifetime calculated from the demodulation.