LAUSR

Luciferase‐based bioluminescence imaging requires no excitation light and thus can be advantageous over fluorescence imaging, but multicolor imaging has been a challenge due to the difficulties of spectral and substrate unmixing. To achieve effective multiplexing of bioluminescence imaging, this study applies phasor analysis that can cluster profiles, in the phasor space, of acquired light intensity according to specific bioluminescence probes. The authors demonstrate that bioluminescence phasor imaging can distinguish heterogeneous cell populations expressing up to six unique reporters. They also resolve two distinct reporters localized to the nucleus and plasma membrane within a single cell. The workflow and optical setup are simple and easily accessible to most cell and developmental biologists, which enables multicolor live‐cell imaging without concerns of photodamage.