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. We characterize and compare imaging performance, longevity, and feasibility of preparations in various configurations. This combination of HG with MPM is highly accessible and opens the doors to new intravital imaging applications.

Internal states involve brain-wide changes that subserve coordinated behavioral and physiological responses for adaptation to changing environments and body states. Investigations of single neurons or small populations have yielded exciting discoveries for the field of neuroscience, but it has been increasingly clear that the encoding of internal states involves the simultaneous representation of multiple different variables in distributed neural ensembles. Thus, an understanding of the representation and regulation of internal states requires capturing large population activity and benefits from approaches that allow for parsing intermingled, genetically defined cell populations. We will explain imaging technologies that permit recording from large populations of single neurons in rodents and the unique capabilities of these technologies in comparison to electrophysiological methods. We will focus on findings for appetitive and aversive states given their high relevance to a wide range of psychiatric disorders and briefly explain how these approaches have been applied to models of psychiatric disease in rodents. We discuss challenges for studying internal states which must be addressed with future studies as well as the therapeutic implications of findings from rodents for improving treatments for psychiatric diseases.