PRIAMOS: A technique for mixing embedding media for freely adjusting pH value and refractive index (RI) for optical clearing (OC) of whole tissue samples

Abstract Investigations of biological samples often require sample transparency, which is achieved by embedding the sample in a high‐refractive index (RI) medium to obtain a homogenous RI distribution in the sample, referred to as optical clearing (OC). Here, we introduce a method for designing embedding media with an increased RI by increasing molecular orbitals, which is achieved by replacing elements in molecules commonly used for OC with elements possessing a more pronounced polarisability. Briefly, we took the established embedding medium Glycerol and exchanged the OH‐groups by Thiol‐groups, resulting in an embedding medium with very similar properties, but with a higher refractive index. We describe a procedure—abbreviated PRIAMOS—to render biological samples transparent using an RI‐matching liquid, which we refer to as p H‐value and R efractive I ndex A djustment by M ixing highly polarisable molecular O rbital S ubstances. We focus on optical clearing in three‐dimensional tissue samples whilst preserving fluorescence of fluorescent labels. The clearing procedure requires 2–3 days, yielding highly transparent samples, preserving the fluorescence of fluorescent proteins like the yellow fluorescent protein (YFP). This is demonstrated on mouse brain samples, imaged with standard confocal microscopy down to 1.6 mm depth, as well as on kidney samples. Mixtures of monothioglycerol, dithioglycerol and tributylamine achieve RI values between 1.52 and 1.57, and an acidity equivalent to pH values between 5 and 8. Our PRIAMOS approach can serve as a guideline for optimising optical clearing protocols.