In frequency domain FLIM the lifetime sensitivity depends on the used modulation frequency. The shorter the expected lifetime, the higher the required modulation frequency to obtain an accurate measurement. A rule of thumb is: \[f = \frac{100}{\tau}.\] With \(f\) in megahertz and \(\tau\) in nanoseconds. For example for GFP transfected cells the lifetime is around 2.5 ns, so the required frequency is about 40 MHz (\(\frac{100}{2.5 ns} = 40\) MHz). Please note that you do not have to change the frequency for slightly different lifetimes, as for a particular frequency the system is sensitive to a broad lifetime range (several nanoseconds). Further note that the above formula is a rule of thumb: it is likely that a frequency slightly different from the rule-of-thumb value gives more accurate results. This is easily verified by measuring at a series of frequencies and inspecting the statistics: the lower the standard deviation, the better the used frequency.