On the Molecular Basis of Shrinkage in Hydrophobic Tissue Clearing
Ali Fakhrolmobasheri, Pushpa Rampratap, Alwin Jansen, Marco van den Noort, Zhongyang Zhang, Bert Poolman, E. van der Giessen, Patrick C.A. van der Wel, P.J. van der ZaagAbstract
Tissue clearing enables the optical imaging of intact tissues and facilitates 3D visualization of complex biological systems and cellular structures. However, tissue clearing using benzyl alcohol-benzyl benzoate (BABB) leads to tissue shrinkage, potentially compromising advanced analysis and applications. The molecular and physical origins of this phenomenon have not been completely understood. To study the molecular aspects of tissue shrinkage, we systematically studied the effects of shrinkage across a range of tissues and process steps. After characterizing the water, lipid, and protein content of these tissues, we found that although tissue clearing does remove lipids, the amount of shrinkage correlates instead with the protein content. Comparative magic-angle spinning solid-state nuclear magnetic resonance (MAS-ssNMR) spectroscopy on tissue undergoing BABB treatment revealed changes in protein conformational dynamics and protein hydration. Combined with analysis of changes in the dielectric constant during the tissue-clearing process, our results indicate that changes in the electrostatic forces between charged molecules (proteins) are more important than lipid extraction for the shrinkage, contrary to what has been hypothesized for shrinkage in tissue clearing.