Expanded applications of bioluminescence microscopy with phasor analysis

Bioluminescence is a powerful technique for monitoring biological processes in vitro and in vivo. A variety of bioluminescent reporters have been developed that span the visible spectrum, but simultaneous detection remains challenging due to substantial spectral overlap. To address this issue, we previously developed bioluminescent phasor analysis to resolve spectrally similar probes. In the current work, we characterize the phasor outputs of several common luciferase reporters and expand the method for distinguishing complex mixtures in tissues. We further apply bioluminescent phasor for imaging biosensors in multidimensional models. Subtle changes in bioluminescent outputs were readily resolved, enabling previously indistinguishable changes in analyte-dependent spectra to be detected. Collectively, this work broadens the utility of bioluminescent phasor imaging for multiplexed imaging applications.