The effect of translational motion on FLIM measurements-single particle phasor-FLIM

Fluorescence lifetime imaging microscopy or FLIM provides a versatile tool for spatially-mapping macromolecular interactions and environments through pixel-by-pixel resolution of the excited-state lifetime. In conventional frequency-domain FLIM the phase and modulation of the detected fluorescence are determined by the photophysics of the fluorophore only. However, translational motion on the timescale of FLIM acquisition can significantly perturb apparent phase and modulation values owing to intensity fluctuations and phase decoherence. Using the phasor plot we outline a simple analytic theory, numerical simulations and measurements on fluorescent beads (ex 470 nm, em 520 nm). Fluctuations due to particle motions result in an increase in the number and spread of phasors, an effect we refer to as phasor broadening. The approach paves the way for the measurement of lifetimes and translational motion from one experiment.