Poster 122 - Optimizing Digestion Protocols to Enhance Optical Coherence Microscopy Resolution Using Expansion Microscopy

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Victor Peng (he/him/his)

High School Student
Eastern Regional High School
Voorhees Township, New Jersey, United States

Co-Author(s)

Fei Wang

Graduate Student
Washington University in St. Louis
University City, Missouri, United States

Primary Investigator(s)

CZ

Chao Zhou

Professor
Washington university in St. louis, United States

Introduction: Examining biological tissues through microscopy has been fundamental to scientific research, offering critical insights into cellular structures and their intricate interactions. However, traditional microscopy techniques, including standard optical and super-resolution methods, often face significant challenges, such as balancing high-resolution imaging with sufficient depth penetration, leading to compromises in speed and cost. Expansion Microscopy (ExM) enhances resolution by physically enlarging samples through the infusion and swelling of a polymer network, achieving near electron microscopy levels of detail[1]. Despite these advantages, using ExM with standard optical microscopes is limited by depth penetration and imaging speed. Optical Coherence Microscopy (OCM) provides a solution by offering high-resolution, three-dimensional imaging deep within tissues[2]. The combination of ExM and OCM, namely Expansion Optical Coherence Microscopy (ExOCM), promises to enhance the visualization of complex biological tissues such as neuronal networks and cancerous tissues. The current study investigates how different digestion times and digestion buffer concentrations affect the resolution of ExOCM, aiming to optimize these parameters for improved imaging quality.

Materials and

Methods: Mouse tissue samples (brain, kidney) were infused with a swellable polymer network solution of acrylamide and sodium acrylate. The samples were first imaged with a spectral-domain OCM (SD-OCM) microscope, then immersed in AcX solution (0.1mg/ml) overnight at room temperature for protein anchoring. After embedding in a gel solution (TEMED, 4-HT, and APS), they were digested by adding 1ml of Proteinase K diluted to 4U/mL or 8U/mL with the digestion buffer in the digestion buffer (50 mM Tris, 25 mM EDTA, 0.5% Triton X-100, 0.8 M NaCl, pH 8.0) and incubated at 60°C for five different time intervals at 30 min, 1 hour, 1.5 hours, 2 hours, and 2.5 hours. Deionized distilled water was added to expand the samples, and they were imaged at 3.5 hours and 24 hours. Simultaneously, tissue samples from the same specimen were cryostat-cut into thin slices, stained with H&E, and imaged with a standard optical microscope, then compared to the images from ExOCM.

Results, Conclusions, and Discussions: Results
The results indicate that the resolution of ExOCM significantly varied with different digestion times and digestion buffer concentrations. Notably, longer digestion times improved imaging quality as digestion times increased from 1 hour to 2.5 hours in 0.5-hour intervals, with 2.5 hours being the optimal digestion time. However, longer digestion times would cause the samples to expand further, but with less visible structures. More structures, such as glomeruli, the cortex and medulla in the kidney, and neurons in the brain, became visible with 2.5 hours of digestion time. For instance, after a 1-hour digestion at 4U Proteinase K concentration (Fig. 2B), brain tissue displayed few visible neurons. With 2-hour digestion at the same concentration (Fig. 2C), more individual neurons became distinct as black spots, and by 2.5 hours (Fig. 2D), fibrous structures were mostly digested, leaving neurons more prominent.
However, expansion had mixed effects. A loss of features in the kidney tissue was observed with protocols using longer digestion times. It is possible that longer digestion times allowed more structural proteins to be degraded, making the samples expand more quickly and possibly too much. This was particularly noticeable after a 3.5-hour expansion, where kidney tissue lost its structure, with glomeruli disappearing (Fig. 3G-H). We are still optimizing the protocol for this sample. In samples treated with a higher concentration of digestion buffer, the entire kidney sample appeared to disintegrate (Fig. 3E-F).

Conclusions
This study demonstrates that optimizing digestion protocols as a part of expansion microscopy is essential for improving the resolution and effectiveness of ExOCM. Tissue type-specific digestion protocols can be beneficial. By fine-tuning these parameters, ExOCM significantly enhances image resolution, allowing for detailed visualization of cellular structures and interactions that are not attainable with traditional microscopy techniques. This advancement provides unprecedented insights, which are particularly valuable for research in neurodegenerative diseases and cancer. Future studies are warranted in further refining these protocols and exploring their application to other complex biological tissues, thus broadening the scope and impact of ExOCM in biomedical research and diagnostics.

Acknowledgements (Optional): My sincere gratitude goes to Professor Zhou for giving me the amazing opportunity to work in his laboratory. I am also grateful to Fei Wang for her guidance and collaboration with me.

References (Optional): 1. Gang Wen, Volker Leen, Taoufik Rohand, Markus Sauer, and Johan Hofkens. Current Progress in Expansion Microscopy: Chemical Strategies and Applications. Chemical Reviews 2023 123 (6), 3299-3323. https://doi.org/10.1021/acs.chemrev.2c00711
2. Bouma, B.E., de Boer, J.F., Huang, D. et al. Optical coherence tomography. Nat Rev Methods Primers 2, 79 (2022). https://doi.org/10.1038/s43586-022-00162-2