Figure 1. Scheme of the double staining and spectral decomposition procedure.

To perform spectral decomposition on live cells labelled with two fluorescent dyes, we first acquired lambda stacks of cells labelled with either Nile Red or laurdan. The emission spectra of the dyes were then extracted from those stacks. Those emission spectra were then used to perform spectral decomposition on the lambda stacks acquired from cells stained with both dyes (bottom right panel).