Endogenous fluorescence lifetime of viable cells by flow cytometry

Autofluorescence emission is commonly measured in flow cytometry and is used as a negative control in protocols that explore binding of exogenous fluorophores to cell receptors or other targets of interest. The presence of intrinsic fluorophores however may burden complex cytometry applications. For example it may be difficult to resolve fluorescence signals from multi-intensity and multi-color measurements when the de-convolved fluorescence in question falls close to the autofluorescence background. One possible solution to intensity and spectral overlap problems in flow cytometry is to acquire fluorescence decay kinetic measurements. To this end we focus on advancing time-dependent flow cytometry and conduct measurements of endogenous fluorescence lifetime. Instrument developments to a phase-sensitive flow cytometry (PSFC) system were coupled with lifetime measurements of intrinsic fluorophores from viable cell samples. The average lifetime of >300,000 individual rat fibroblast cells was measured at discrete wavelengths ranging from 457- to 785-nm using a 10-MHz intensity-modulated excitation beam. AC amplitude, DC, and phase-shift were resolved and the average lifetime from excitable endogenous species was measured. The lifetime results ranged from 1- to 6-ns over the broad spectral range. Cataloging lifetime values prefaces the use of phase-sensitive techniques in more complex systems and provides a priori measurements necessary for PSFC filtering known lifetime signals from Raman, or other emission and scattering events.