Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection

Fluorescence correlation spectroscopy (FCS) is a powerful technique to meas ure chemical reaction rates and diffusion coefficients of molecules in ther mal equilibrium. The capabilities of FCS can be enhanced by measuring the e nergy, polarization, or delay time between absorption and emission of the c ollected fluorescence photons in addition to their arrival times. This info rmation can be used to change the relative intensities of multiple fluoresc ent species in FCS measurements and, thus, the amplitude of the intensity a utocorrelation function. Here we demonstrate this strategy using lifetime g ating in FCS experiments. Using pulsed laser excitation and laser-synchroni zed Elating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating te chnique are presented: suppression of background fluorescence, simplificati on of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for me asuring forward and backward rates of protein fluctuations in equilibrium a nd for distinguishing between static and dynamic heterogeneity makes it a p romising tool in the investigation of chemical reactions and conformational fluctuations in biomolecules.