Label-Free Three-Dimensional Morphological Analysis of Live Organoids Using Low-Coherent Holotomography

Three-dimensional (3D) visualization and morphological dissection of organoids is challenging due to the thickness and its complex structure. Fluorescence-based imaging methods have been widely used; however, those are not suitable for observing morphological changes of growing organoids without damaging the samples. Holotomography (HT) has emerged as a useful tool for imaging live specimens without additional pre-treatment such as fixation, fluorescence labeling and excitation. HT can achieve long-term 3D observation of live specimens for weeks without phototoxicity. Furthermore, the measured individual cell data can be analyzed to elucidate not only the spatio-temporal 3D dynamics with high spatial resolution, but also the quantitative information of its subcellular compartments such as volume and dry mass.

In this study, we applied a recently developed low-coherent holotomography imaging system, HT-X1, which provides label-free 3D visualization of live cells and multicellular specimens. Using the HT-X1, we successfully observed in situ 3D morphological dynamics of patient-derived human lung organoids with Matrigel-embedded condition. The location and its thickness of the mucosal layer inside of which has a lower refractive index (RI) were also detected, since HT enables the volumetric reconstruction of RI which provides quantitative morphology analysis with unprecedented precision and resolution. The other subcellular features such as nuclei, nucleoli, mitochondria, lipid droplets, chromosomes, and multiciliate structures were observed with the high resolution of the HT-X1. Throughout the results, we suggest that low-coherent holotomography has an exclusive capability for phenotypic and quantitative studies of diverse types of organoids and other 3D multicellular live specimens including stem cells.