ClearGel: a thermoresponsive hydrogel for chronic, near-millimeter two-photon imaging through the intact skull

The intact skull remains a fundamental barrier to high-resolution two-photon neuroimaging in live mice. Existing skull-clearing methods, however, cannot simultaneously provide deep cortical access, spatial selectivity, reversibility, and biological safety. Here we introduce ClearGel, a thermoresponsive hydrogel that undergoes rapid in situ gelation upon contact with the skull, confining the clearing agents to the targeted region while sustaining their release over time. ClearGel routinely enables two-photon imaging down to approximately 800 μm through the intact skull-a depth statistically equivalent to open-skull windows and substantially surpassing all prior approaches. It supports user-defined clearing patterns from focal sites to whole-skull coverage, allows repeated imaging over 14 days through a reversible optical interface, and, critically, enables awake functional imaging of both neuronal Ca2+ activity and the more elusive cAMP dynamics at single-cell resolution. ClearGel requires only simple topical application-no craniotomy or skull thinning-and provides a minimally invasive, chronic-compatible optical window for longitudinal studies of cortical function in behaving mice.