Label-Free 3D High-resolution Analysis of Cellular Differentiation in Live Organoids Using Low-Coherence Holotomography

Session

Advances in Label-free Imaging

Authors

Jaehyeok Lee (3), Hye-Jin Kim (3), Mahn Jae Lee (2), Geon Kim (2), Bon-Kyoung Koo (1), Sumin Lee (3), Prof. YongKeun Park (2)

Affiliations

1. IBS
2. KAIST
3. Tomocube

Keywords

quantitative phase imaging; 3D; microscopy; label-free; refractive index; holotomography; organoid

Abstract text

Three-dimensional (3D) visualization is essential for understanding the physiological functions and cell type diversity of live organoids. However, conventional 3D imaging techniques based on fluorescence markers have limitations for long-term, non-invasive observation of growing organoids, as they require exogenous manipulations such as immunostaining, genetic modifications, or fluorophore excitations. Holotomography offers a potential solution by providing high-resolution 3D imaging of live, unlabeled organoids. Holotomography exploits the refractive index (RI) of a sample as an intrinsic imaging contrast, and RI information can also be translated into quantitative analyses of biomolecular concentration or volumetric analysis of subcellular organelles.

Here, we employed a low-coherence holotomography imaging system suitable for observing multicellular specimens to examine the morphological features of early-stage murine small intestine organoids. We acquired 3D RI tomograms of live organoids embedded in Matrigel for 120 hours, revealing the early differentiation of small intestine organoids, including the formation of a central cyst structure and crypt-like budding structures. The differentiation of enterocytes, goblet cells, and Paneth cells was distinctly identified through the marker-free observation of subcellular structures such as secretory vesicles and granular structures. Furthermore, we observed cellular dynamics, such as mitotic cell division and the translocation and chromatin condensation of apoptotic cells. We also analyzed the volume and dry mass of individual organoids, exhibiting fluctuations due to size oscillation and organoid fusion.

In conclusion, low-coherence holotomography provides unique capabilities for determining the differentiation qualities of organoids, making it a valuable tool for basic research and therapeutic applications. By enabling non-invasive, label-free, and long-term observation of live organoids, this technology has the potential to revolutionize the field of organoid research. This study offers important insights into the early stages of murine small intestine organoid differentiation, demonstrating the potential of holotomography as a powerful tool for investigating the development and function of organoids.

References