Luminescence lifetime imaging integrated into selective plane illumination microscopy for oxygen imaging in 3D cell cultures.

There is a growing need for 3D imaging that records not only cellular structures but also key chemical parameters, such as local oxygen tension or pH within 3D cell cultures. To meet this need, we have designed and developed a 3D luminescence microscopy platform that combines two microscopy modalities: selective plane illumination microscopy (SPIM) and luminescence lifetime imaging microscopy (LLIM). The SPIM-LLIM combination enables rapid 3D imaging with low phototoxicity and versatile use of chemical indicators responsive in luminescence lifetime. Here, we report on the design, development, and characteristics of the platform when applied to phosphorescence lifetime imaging microscopy (PLIM)-based oxygen imaging. Using commercial oxygen-sensitive microparticles, we show that camera-based frequency-domain PLIM in the kHz range, combined with a straightforward intensity-threshold preprocessing, allows for sensitive and robust 3D oxygen imaging at physiological oxygen levels. As a demonstration, we apply the system to oxygen imaging of 3D cardiac constructs with phosphorescent extra- and intracellular oxygen indicators. The results show that the oxygen tension in the core of the constructs often becomes compromised or even hypoxic. Furthermore, we show that the acquired oxygen tension profiles are consistent with diffusion-reaction models and can provide useful estimates for the oxygen consumption rates inside the cellular constructs. The platform offers a wide application potential in 3D