(Invited) Rapid Optical Tissue Clearing Using Polyelectrolyte Hydrogels for Three-Dimensional Fluorescence Imaging

Abstract

Introduction: Optical tissue clearing methods have been attractive for three-dimensional fluorescence imaging of intact tissues and organs. Most tissues are optically opaque, because tissue components such as water, protein and lipids have different refractive indices. A CLARITY method using polyacrylamide gels was recently developed. There are four steps in this method to achieve the optical tissue clearing. (1) Tissues containing chemical reagents are prepared, (2) proteins are embedded in the hydrogel, (3) lipids are removed from the tissue, and (4) the refractive index of media is adjusted. Brains became easily transparent using this method, but extracellular matrix (ECM)-rich tumor tissues did not. The third step is a rate-limiting step. We attempted to apply polyelectrolyte hydrogels to the CLARITY method for rapid removal of lipid molecules. Polyelectrolyte hydrogels are highly swollen, as the polymer networks expand because of the electrostatic repulsion between charged groups on the polymer chains. Thus, it was expected that polyelectrolyte hydrogels would contribute to rapid optical clearing because of the rapid removal of lipid molecules. Experiments: Sodium styrenesulfonate (SS), sodium 2-acrylamido-2-methylpropane sulfonate (AMPS) and acrylic acid (AcA) were used as anionic monomers. Hydrogels were prepared by using acrylamide (AAm), anionic monomers, a water soluble azo initiator (VA-044), in the presence of bis-methyleneacrylamide (bisAA). Optical tissue clearing was performed, as follows. The tissues were immersed in 10 mL of phosphate buffer saline (PBS) solution (pH 7.4) containing monomers (5.6 mmol in total), bisAA (50 mg), VA-044 (125 mg) and paraformaldehyde (400 mg) at 4oC for 24 h. Then, the hydrogel was prepared at 37oC for 24 h. After removing the gel outside the tissue, tissues were immersed in 30 mL of 0.8 M boric acid buffer containing 4% sodium dodecyl sulfate (SDS) at 37oC and were shaken for 3 days. Photos of the tissues were taken before and after the SDS treatment. Then, the tissue was placed in 30 mL of 0.1% (vol/vol) of a Triton X-100 solution. After shaking at 37oC for 2 days, it was immersed in 20 mL of ethylene glycol for 1 h. Photos of the tissues were then taken. Results and Discussion: First, a poly(AAm-co- SS) hydrogel was designed as a polyelectrolyte hydrogel for the rapid optical tissue clearing. Although poly(sodium styrenesulfonate) hydrogels were not prepared, the poly(AAm-co-SS) hydrogels with not more than 80mol% of SS could be prepared. The swelling ratio of the poly(AAm-co-SS) hydrogels became higher with a higher SS content, as expected. The optical clearing of ECM-rich tumor tissues with 15 mm × 10 mm × 1 mm was performed. The optical tissue clearing proceeded more rapidly, by using poly(AAm-co-SS) hydrogel at higher SS content. It took 2 months to clear tumor tissues using the polyacrylamide hydrogel, but only 8 days using the poly(AAm-co-SS) hydrogel at a ratio of 25/75, as shown in the attached image. Next, polyelectrolyte hydrogels with AMPS and AcA were prepared to compare the physicochemical properties and the speed of optical tissue clearing of various tissues. These polyelectrolyte hydrogels were highly swollen, and the poly(AAm-co-AMPS) hydrogel became larger than the poly(AAm-co-SS) and poly(AAm-co-AcA) hydrogels even at the same mole content. It may be because AMPS is a hydrophilic and strong electrolyte. The tumor tissues embedded in these polyelectrolyte hydrogels became transparent more rapidly, compared with the conventional method using a polyacrylamide gel. However, the clearing speeds of brain were similar among these hydrogels with and without anionic monomers. The swelling behaviors of brain tissues were different. Brain tissues embedded in the poly(AAm-co-AMPS) and poly(AAm-co-SS) hydrogels were swollen, but not in the poly(AAm) and poly(AAm-co-AcA) hydrogels. The fluorescence imaging was performed by using green fluorescent protein (GFP)-expressing tissues. The fluorescence signals were observed from tissues embedded in the poly(AAm-co-AcA) hydrogel with a similar level to those in the polyacrylamide hydrogel. However, the fluorescence signals from tissues embedded in the poly(AAm-co-AMPS) and poly(AAm-co-SS) hydrogels were diminished, suggesting that GFP in tissues was denatured by these polymers. Conclusions: The poly(AAm-co-AcA) hydrogels were the best polyelectrolyte hydrogel in this study for optical tissue clearing and the subsequent fluorescence imaging, because the clearing speed was higher and the polymer did not affect the proteins very much. Figure 1