Fluorochrome separation by fluorescence lifetime phasor analysis in confocal and STED microscopy

In fluorescence microscopy, discrimination of fluorochromes in multi-color labeling was originally based on the emission spectrum only, then on emission and distinct excitation wavelengths. With the advent of faster and easier to use fluorescence lifetime imaging microscope (FLIM) systems, an additional, third level of discriminating fluorochromes becomes feasible. In this tutorial, we describe how to separate two fluorochromes, one with shorter and one with longer fluorescence lifetime, in a single spectral channel. The separation is done with the help of a phasor diagram of the lifetime information. We applied the method on images made by confocal or stimulated emission depletion (FLIM-STED) microscopy but it is transferable to other FLIM methods. This approach works with considerable less photons than separation by curve fitting. Images can be recorded at speeds comparable to normal confocal or STED microscopy. One shown example has two spectral channels with two fluorochromes each, plus another neighboring color channel in which spectral bleed-through and reflection is corrected by lifetime properties. All fluorochromes as well as the hard- and software used are commercially available. Lifetime separation generally may double the number of fluorochromes that can be used in fluorescence microscopy.