ABSTRACTTissue clearing techniques are undergoing a renaissance motivated by the need to image fluorescence deep in biological samples without physical sectioning. Optical transparency is achieved by equilibrating tissues with high refractive index (RI) solutions, which require expensive optimized objectives to avoid aberrations. One may thus need to assess whether an available objective is suitable for a specific clearing solution, or the impact on imaging of small mismatches between cleared sample and objective design RIs. We derived closed form approximations for image quality degradation versus RI mismatch and other parameters available to the microscopist. We validated them with computed (and experimentally confirmed) aberrated point spread functions, and by imaging fluorescent neurons in high RI solutions. Crucially, we propose two simple numerical criteria to establish: (i) the degradation in image quality (brightness and resolution) from optimal conditions of any clearing solution/objective combination; (ii) which objective, among several, achieves the highest resolution in a given immersion medium. These criteria apply directly to the widefield fluorescent microscope but are also closely relevant to more advanced microscopes.
Two simple criteria to estimate an objective’s performance when imaging in non design tissue clearing solutions