Determining the Depth Limit of Bioluminescent Sources in Scattering Media

Bioluminescence has several potential advantages compared to fluorescence microscopy for in vivo biological imaging. Because bioluminescence does not require excitation light, imaging can be performed for extended periods of time without phototoxicity or photobleaching, and optical systems can be smaller, simpler, and lighter. Eliminating the need for excitation light may also affect how deeply one can image in scattering biological tissue, but the imaging depth limits for bioluminescence have yet to be reported. Here, we perform a theoretical study of the depth limits of bioluminescence microscopy and find that cellular resolution imaging should be possible at a depth of 5-10 mean free paths (MFPs). This limit is deeper than the depth limit for confocal microscopy and slightly lower than the imaging limit expected for two-photon microscopy under similar conditions. We also validate our predictions experimentally using tissue phantoms. Overall we show that with advancements in the brightness of bioluminescent indicators, it should be possible to achieve deep, long-term imaging in biological tissue with cellular resolution.