PRIAMOS: A technique for mixing embedding media for freely adjusting pH value and refractive index (RI) for optical clearing (OC) of whole tissue samples.
Ulrich Leischner, Martin Reifarth, Monika S Brill, Florian Schmitt, Stephanie Hoeppener, David Unnersjö Jess, Hjalmar Brismar, Ulrich S Schubert, Rainer Heintzmann
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引用次数: 0
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 pH-value and Refractive Index Adjustment by Mixing highly polarisable molecular Orbital Substances. 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.
期刊介绍:
The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.
The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.
Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.