Rapid increase in transparency of biological organs by matching refractive index of medium to cell membrane using phosphoric acid

Tissue clearing is a fundamental challenge in biology and medicine to achieve high-resolution optical imaging of tissues deep inside intact organs. The clearing methods reported up to now require long incubation times or physical/electrical pressure to achieve tissue clearing, which is done by matching the refractive indices of the whole sample and medium to that of the lipid layer. Here we show that phosphoric acid increases the refractive index of the medium and can increase the transparency of formalin-fixed tissue samples rapidly. While phosphoric acid (8.5-14.2 M) suppresses bright signals on the boundary of cells in their phase-contrast images, it does not damage the morphology of the phospholipid cell membrane. Immersion of fixed tissues of mice in phosphoric acid solutions (8.5-14.2 M) increased their transparency within 60 min in the case of 3 mm-thick fixed tissue specimens. Although further investigations are needed to apply this protocol to three-dimensional fluorescence imaging or immunohistochemistry, the protocol presented herein may contribute to developing better and faster soaking methods for tissue clearing than previously reported protocols. Tissue clearing effect of phosphoric acid is fast and needs only 60 min incubation.