Choosing a skull clearing technique for chronic mesoscopic optical imaging in awake mice.

Significance: Transcranial imaging windows are crucial for Aim: We aimed to methodically compare and modify current topical skull optical clearing methods in search of an optimal technique for chronic awake widefield imaging. Approach: We used widefield intrinsic optical-signal imaging and laser speckle contrast imaging to investigate the applicability of these methods. Results: Application of a UV-curable optical adhesive, NOA61 both as a monotreatment and in combination with EDTA, proved suitable for long-term awake mesoscopic imaging, providing long-term global transparency. The previously described Triple-S method led to inflammatory-like reaction and/or opacification of the window over subsequent days. Cyanoacrylate or methyl-methacrylate (Metabond-equivalent) treatments, on the other hand, led to drying artifacts, occluding parts of the window. Therefore, these latter methods made awake bihemispheric imaging impractical. EDTA-NOA61 combination was then tested for documenting interhemispheric connectivity changes following optogenetic triggering of cortical spreading depolarizations. Conclusions: We discussed the advantages and drawbacks of previous skull clearing methods and identified suitable methods for awake mesoscopic chronic imaging. These findings highlight the importance of minimally invasive skull optical clearing methods for widefield use in awake mice.