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.
This content will become publicly available on June 27, 2025
DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and non‐equilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high‐throughput screening. To address this, recent advancements in bioreactor‐based plasmid DNA production is coupled with anion exchange chromatography to produce a unified approach to generating gram‐scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL−1of uniform supercoiled and relaxed circular plasmid DNA, which is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements on DNA of different length, topologies, and concentrations at sample volumes up to 1 mL. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time‐concentration superposition curve that spans twelve decades of frequency. Ultimately, these results could provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.
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more » « less- PAR ID:
- 10519353
- Publisher / Repository:
- Wiley
- Date Published:
- Journal Name:
- Advanced Materials
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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