Molecular signals interact to mediate diverse biological computations. Ideally one would be able to image many signals at once, in the same living cell, to reveal how they work together. Here we report temporally multiplexed imaging (TMI), which uses the clocklike properties of fluorescent proteins to enable different cellular signals to be represented by different temporal fluorescence codes. Using different photoswitchable fluorescent proteins to represent different cellular signals, we can linearly decompose a brief movie of the fluorescence fluctuations in a given cell, into a sum of the fluctuation traces of each individual fluorophore, each weighted by its respective signal amplitude. We demonstrate the power of TMI to report relationships amongst a diversity of second messenger, kinase, and cell cycle signals, using ordinary microscopes.
