ABSTRACT
For frequency-domain FLIM (fluorescence lifetime imaging microscopy), the amplitude of the excitation light is modulated repetitively at high frequency (HF). Radio frequencies between 1 and 200 MHz are nominal and chosen in order for the fluorescence response (nanoseconds) to be sensitive to the frequency of repetition/modulation. The waveform of the HF modulation is often sinusoidal, but it can be any repetitive shape; such a repetitive waveform can be decomposed into multiple harmonics in a Fourier series, and each sinusoidal harmonic component is treated separately. Only the fundamental frequency is present if the excitation is a pure sinusoidal modulation; other waveforms (e.g., square waves or pulse trains from mode-locked lasers) contain multiple harmonics of the repetition frequency.
