Evaluation of skull optical clearing process for longitudinal non invasive optical imaging

We have evaluated in vitro and in vivo the efficiency and practicability of reversible optical clearing of the skull for minimally invasive, longitudinal imaging of the rodent brain. Firstly, in vitro experiments have been conducted on resected mice skulls to evaluate the efficiency of the optical clarification process using different optical clearing agents. On the basis of recent literature, we have evaluated in vitro different optical clearing processes: (i) the direct application of PEG400; (ii) sequential EDTA and glycerol application and (iii) application of a solution of urea dissolved in ethanol. First, the time course of the clarification of the skull has been monitored quantitatively. We have carried out photometry experiments at 633 nm using a two integrating spheres system to characterize the total transmittance and reflectance of the mice skull samples. The evaluation of the optical transmission coefficient at 1300 nm was also obtained at different time points of the clearing process using sequential optical coherent tomography (OCT) imaging of the skull samples during the clearing process. Second, in vivo evaluation was carried out for repeated transcranial mapping of brain blood flow after optical clarification of the skull. Relative blood flow maps were obtained from multiple exposure laser speckle imaging. Third, post-mortem analysis of the toxicity of the chemicalstopical application was evaluated using immunohistochemistry to asses eventual cells death and inflammation. Overall our results show that in vivo brain imaging in mice could benefit from in vivo optical clearing. Yet, the use of optical clearing agents in vivo requires a proper evaluation of their efficiency, practicability of their use and potential toxicity to the tissues, especially for long term longitudinal studies.