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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I9 - Optimization of imaging chambers for single-cell resolution time-lapse imaging of whole organs

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Nneka Onyea (she/her/hers)

Undergraduate Research Assistant in Biological Engineering
Princeton University
Los Angeles, California, United States

Co-Author(s)

Chan Jin Park, PhD

Postdoctoral research associate
Princeton University
Princeton, New Jersey, United States

Primary Investigator(s)

Celeste M. Nelson, PhD (she/her/hers)

Wilke Family Professor in Bioengineering
Princeton University
Princeton, New Jersey, United States

Introduction: Confocal fluorescence microscopy can provide valuable insights into the architecture of
multicellular tissues, often at cellular or subcellular resolution. Imaging of fixed samples yields information about tissues at snapshots in time, but sacrifices the ability to observe temporal changes that accompany tissue development and remodeling. Conversely, time-lapse imaging yields information about how cells rearrange within tissues over time, but conditions that maintain the viability of samples are often incompatible with achieving cellular or subcellular spatial resolution. For example, the most common platform for imaging of embryonic organs is to culture the sample at the air-liquid interface using a porous membrane. However, this porous membrane obstructs light, resulting in blurry, low-resolution images and videos. To overcome this challenge, we created an optimized live-imaging chamber using tissue-culture plates and transparent polyacrylamide gels to increase spatial resolution of time-lapse imaging while maintaining sample viability. As a proof-of-concept, we used this approach to visualize embryonic lungs with single-cell resolution, which revealed novel rearrangements within the epithelium of the lung over developmental time.

Materials and

Methods: Imaging chambers were prepared by drilling a 25-mm hole into the bottom of a 6-well culture plate. A treated glass coverslip was secured to the bottom of the plate using VPS duplicating materials and cured for 15 minutes at 65°C. Acrylamide was polymerized on the surface of the coverslip, then submerged in culture medium for 1 hour.

Fertilized chicken (Gallus gallus) eggs were incubated at 38°C in a humidified atmosphere for five days. Lungs from embryos at embryonic day 5 (E5) were microdissected and then were placed on top of the polyacrylamide gel in the imaging chamber. To visualize individual cells, Cellbrite Steady 650 membrane dye was injected into the lumen of the lung explants. Samples were visualized via time-lapse imaging using both confocal point scanning and two- photon microscopes.

Results, Conclusions, and Discussions: When cultured within this novel imaging chamber, embryonic chicken lung explants survive and undergo branching morphogenesis, which we visualized at single-cell resolution using the membrane dye. Two-photon microscopy revealed that the lung epithelium is primarily columnar during morphogenesis, but that individual cells round up at the apical surface prior to mitosis. Because we could achieve both cellular and temporal resolution, we were able to observe the angles of these divisions, which appear to depend on the behaviors of neighboring epithelial cells. Future studies will track the positions of epithelial cells over developmental time as a function of location and interactions with their surrounding microenvironment. We believe that this platform could be used for time-lapse imaging of a variety of tissue samples.

Acknowledgements (Optional): We thank the Tissue Morphodynamics Lab at Princeton University!