Transport in the brain studied with in vivo two-photon microscopy: the impact of spatial and temporal resolution

Understanding molecular transport in the brain in vivo is essential for elucidating how the brain regulates its metabolism, how neurological pathologies develop, and why many brain-targeted drugs fail. Two-photon microscopy (TPM) is the gold standard for in vivo imaging in highly scattering tissues such as the brain. However, suboptimal use of TPM can compromise study outcomes due to the inherent challenges of in vivo imaging. We highlight the importance of optimizing both spatial and temporal resolution in TPM to ensure accurate data acquisition and interpretation. We compare TPM-based studies of molecular transport with traditional wide-field microscopy approaches, emphasizing how light scattering in brain tissue limits the effectiveness of the latter. We discuss the impact of motion blur-arising from diffusion of tracers or natural movement of cerebral vasculature-on image quality and offer practical strategies to mitigate these effects. In addition, we address the complexities of statistically analyzing noisy images, typically occurring due to low-photon budgets or the need for fast image recording in in vivo TPM. We conclude with a set of practical guidelines for effective data acquisition, aimed at facilitating the implementation of the concepts discussed. When properly optimized, TPM is a powerful tool capable of revealing fundamental mechanisms of brain transport and advancing our understanding of cerebral metabolism.