Investigating cellular dynamics of live organoids via label-free 3D high-resolution analysis with low-coherence holotomography

Session

Poster Session

Authors

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

Affiliations

1. Institute for Basic Science (IBS)
2. Korea Advanced Institute of Science and Technology (KAIST)
3. Tomocube

Keywords

Holotomography, Label-free imaging, Organoid imaging, Image analysis, Long-term mornitoring

Abstract text

Three-dimensional (3D) visualization is essential for understanding the physiological functions and cell type diversity of live organoids. But conventional 3D imaging techniques based on fluorescence markers have limitations when it comes to long-term, non-invasive observation of live organoids. Holotomography takes advantage of a non-invasive imaging technique since it exploits the refractive index (RI) of a sample as an intrinsic imaging contrast. The RI information can also be translated into quantitative analyses of biomolecular concentration or volumetric analysis of subcellular organelles.

In this study, we employed a low-coherence holotomography imaging system, which is suitable for observing multicellular specimens, to examine the morphological features of murine small intestine organoids (sIOs). With the low-coherence holotomography system, we were able to monitor undisguised live organoids just as they are. We acquired 3D RI tomograms of live organoids embedded in Matrigel for 120 hours and revealed the early differentiation of sIOs, 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. Also, we explored the drug response of sIOs by the Cisplatin treatment. We were able to track the shrinkage of crypt due to the cell death, and translocation of a single apoptotic cell.

In conclusion, low-coherence holotomography provides unique capabilities for determining the status of development, differentiation, and viability 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.

References

Rios, A.C. & Clevers, H. Imaging organoids: a bright future ahead. Nature methods 15, 24–26 (2018).

Park, Y., Depeursinge, C. & Popescu, G. Quantitative phase imaging in biomedicine. Nature photonics 12, 578–589 (2018).

Hugonnet, H., Lee, M. & Park, Y. Optimizing illumination in three-dimensional deconvolution microscopy for accurate refractive index tomography. Opt. Express 29, 6293–6301 (2021).