Enhanced flavoprotein autofluorescence imaging in rats using a combination of thin skull window and skull-clearing reagents

Flavoprotein autofluorescence (FA) imaging is a powerful technique for investigating neural activity in vivo. However, its application in rats is limited by the thickness of the intact skull, which reduces light transmission and signal-to-noise ratio (SNR). In this study, we introduce a novel approach that integrates a thin skull window (TSW) with a skull-clearing reagent (CTSW) to enhance FA imaging in rats. The FA signals evoked by somatosensory stimulation were recorded under both TSW and CTSW conditions. The results demonstrate that CTSW significantly improved the SNR of FA signals compared to TSW alone, enabling more precise detection of neural activity. Notably, the enhanced signal clarity facilitated robust imaging in the secondary motor cortex (M2), a region where activity is barely detectable using conventional TSW. By better preserving intracranial physiological conditions than craniotomy, CTSW minimizes postoperative complications and supports longitudinal imaging. Furthermore, this technique may be applicable to other optical imaging modalities, including calcium and vascular imaging. The ability to enhance cortical signal detection while maintaining a minimally invasive preparation positions CTSW as a promising tool for functional mapping and long-term studies in behaving rats.