Search for: All records

CLARITY is a tissue preservation and optical clearing technique whereby a hydrogel is formed directly within the architectural confines of ex vivo brain tissue. In this work, the extent of polymer gel formation and crosslinking within tissue was assessed using Raman spectroscopy and rheology on CLARITY samples prepared with a range of acrylamide monomer (AAm) concentrations (1%, 4%, 8%, 12% w/v). Raman spectroscopy of individual neurons within hybrids revealed the chemical presence and distribution of polyacrylamide within the mouse hippocampus. Consistent with rheological measurements, lower %AAm concentration decreased shear elastic modulus G’, providing a practical correlation with sample permeability and protein retention. Permeability of F(ab)’2 secondary fluorescent antibody changes from 9.3 to 1.4 µm² s⁻¹going from 1 to 12%. Notably, protein retention increased linearly relative to standard PFA-fixed tissue from 96.6% when AAm concentration exceeded 1%, with 12% AAm samples retaining up to ~ 99.3% native protein. This suggests that though 1% AAm offers high permeability, additional %AAm may be required to enhance protein. Our quantitative results on polymer distribution, stability, protein retention, and macromolecule permeability can be used to guide the design of future CLARITY-based tissue-clearing solutions, and establish protocols for characterization of novel tissue-polymer hybrid biomaterials using chemical spectroscopy and rheology.

 

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type–specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.