Genetically-encoded fluorescence-based sensors have emerged as an essential tool for measuring the abundance of heme, revealing its trafficking pathways, and probing its signalling and regulatory role in cells. A number of different sensor designs have been described in the literature, and these typically report on the abundance of exchangeable heme via an intensity modulation of the emission from fluorescent-protein reporters. Here, we show that multi-photon fluorescence-lifetime imaging microscopy (MP-FLIM) can be used to monitor the response of heme sensors in transfected-HEK293 cells. The adoption of a multi-photon approach could extend heme quantification further to deep-tissue imaging in the future, where it could also reduce phototoxicity as the non-linear excitation of fluorescent reporters is confined to the focal volume
