Multiphoton microscopy (MPM) has become a preferred technique for intravital imaging deep in living tissues with subcellular detail, where resolution and working depths are typically optimized utilizing high numerical aperture, water-immersion objectives with long focusing distances. However, this approach requires the maintenance of water between the specimen and the objective lens, which can be challenging or impossible for many intravital preparations with complex tissues and spatial arrangements. We introduce the novel use of cohesive hyaluronan gel (HG) as an immersion medium that can be used in place of water within existing optical setups to enable multiphoton imaging with equivalent quality and far superior stability.
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