Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session D

Poster G2 - Preservation and deep multiphoton imaging of developing organs via solid state acrylamide clearing

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  A major challenge in biological research is the ability to clear and preserve large tissue specimens for imaging. Common protocols employ solvent-based clearing solutions. However, solvent evaporation and gradual photobleaching decay the quality of precious immunostained samples. Here we implement a recently published solid-state (solvent free) clearing method using a high-n acrylamide copolymer to clear and preserve the sample. We apply this method to image large (thicker than 1 mm) immunostained whole mouse embryos (E10.5) and quantify the depth of clearing against BA:BB clearing and the comparative resilience to photodamage by high laser exposure. We then use this volume-preserving clearing method to create a series of confocal images of the chicken lung from embryonic day 5 to 10. Finally, we present a workflow to quantify the epithelial architecture of the chicken lung.

Materials and

Methods:  To prepare the acrylamide monomer solution, we followed the protocol established by Hsiao et al., "Transparent tissue in solid state for solvent-free and antifade 3D imaging" (2023).

We collected embryonic day 10.5 mouse embryos, and chicken lungs dissected from embryonic day 5 to 10. Tissue was fixed in 4% Paraformaldehyde for 30 minutes. Then the tissue was permeabilized using 0.5% TBS-Triton X (TBST). Prior to immunostaining, the tissue was blocked using 10% Donkey Serum in TBST. Primaries, Tom20 and E-Cadherin, were incubated in the blocking buffer for two days at 4°C. Secondaries, Alexa Fluor 555 and Alexa Fluor 680 along with DAPI, were incubated in the blocking buffer for 24 hours.

After staining, the tissue was dehydrated in a methanol series (25%-50%-75%-100%). Half of the mouse embryos were then cleared in BA:BB and half in the acrylamide copolymer. Acrylamide samples were then rehydrated through a methanol series (25%-50%-75%-PBS). The samples were washed in acrylamide monomer solution for 45 minutes and then again for 90 minutes. The samples were then moved to a desiccator for an hour. The sample was then transferred to an imaging chamber with a mm spacer with fresh monomer solution. The acrylamide was cured with 360 nm UV at 3.06 mW/cm2 in 90-second increments to avoid overheating for a total of approximately 15 minutes.

Results, Conclusions, and Discussions: The acrylamide copolymer qualitatively cleared tissue more than its BA:BB alternative. To quantify the relative clearing quality of the acrylamide method, the samples were confocally imaged on both traditional scanning and 2-photon confocal microscopes.

We quantified the intensity decay of imaging along the depth of the sample. The decay rate of the intensity was considerably less in the acrylamide sample, with the intensity decaying with an exponent of 7.8E-4 1/micron for the acrylamide sample and an exponent of 1.7E-3 1/micron for the BA:BB sample. This is promising because it allows for larger samples to be imaged deeper with minimal change in intensity due to scattering. Furthermore, since the tissue is embedded in a solid, with glass on both sides, the sample can be flipped on a traditional confocal microscope to image from both sides, a technique that is often unrealistic in a solvent-cleared tissue.

Using the high 2-photon laser flux, we compared the effects of photobleaching and photodamage on the BA:BB cleared samples versus the acrylamide copolymer cleared samples. After 5 minutes of continuous imaging from a 350 mW 2-photon laser at 850 nm, the BA:BB cleared sample’s intensity of Alexa 555 was reduced by approximately 17% while the acrylamide copolymer cleared sample only lost approximately 3% of its intensity. This drastic difference shows the acrylamide copolymer helps to protect the fluorophores from photobleaching. Additionally, the BA:BB cleared sample was immediately damaged from microcavitation upon exposure to laser at 1000 mW. The acrylamide sample was exposed to up to 3500 mW power and did not show any signs of microcavitation.

Having quantified the clearing technique for large tissues, we generated a proof-of-concept dataset using the branching chicken lung from embryonic day 5 to embryonic day 10. We created a protocol to stain, clear, and confocally image the lungs. We then created a procedure to segment the epithelium from the mesenchyme, and to numerically quantify the volume and surface area of the lungs.

This solid-state clearing method shows promise for improved imaging of large tissue specimens as well as preserving the samples for repeated use.

Acknowledgements (Optional):  This project was supported by the NSF and the Princeton Center for Complex Materials an NSF-supported MRSEC.