Event Ordering in Live-Cell Imaging Determined from Temporal Cross-Correlation Asymmetry

We use the temporal asymmetry of the cross-correlation function to determine the temporal ordering of spatially localized cellular events in live-cell multichannel fluorescence imaging. The analysis is well suited to noisy, stochastic systems where the temporal order may not be apparent in the raw data. The approach is applicable to any biochemical reaction not in chemical equilibrium, including protein complex assembly, sequential enzymatic processes, gene regulation, and other cellular signaling events. As an automated quantitative measure, this approach allows the data to be readily interpreted statistically with minimal subjective biases. We first test the technique using simulations of simple biophysical models with a definite temporal ordering. We then demonstrate the approach by extracting the temporal ordering of three proteins—actin, sorting nexin 9, and clathrin—in the endocytic pathway.