Fluorescence lifetime unmixing enables the independent imaging of two or three fluorophores that have identical emission spectra. The separation is based on differences in the excited state lifetime. This imaging method is of interest as it increases the options for multiplex imaging. (It aligns with my talk on the engineering of fluorescent proteins). We have engineered fluorescent proteins by screening for lifetime variants (https://doi.org/10.1038/nmeth.1415). As a result, we have identified and characterized fluorescent proteins with different fluorescence lifetimes and have demonstrated that these can be used for lifetime unmixing (https://doi.org/10.1529/biophysj.107.125229). In this workshop, we will discuss the requirements for lifetime unmixing and the types of fluorescent probes that are available. A sample with two different Cyan Fluorescent Proteins (CFPs) that are targeted to two different cellular locations will be used (https://doi.org/10.1111/jmi.12168). The conditions for imaging and analysis will be discussed and the participants will have the opportunity to do the acquisition and tweak imaging conditions. The workshop has a focus on the data acquisition, but aims to explain and show the data analysis&visualization (using the software of the microscopy setup) as well. The outcome of the experiment is a set of two images with the distribution of the two cyan fluorescent proteins.