Improved 3D cellular morphometry of Caenorhabditis elegans embryos using a refractive index matching medium

Cell shape change is one of the driving forces of animal morphogenesis, and the model organism Caenorhabditis elegans has played a significant role in analyzing the underlying mechanisms involved. The analysis of cell shape change requires quantification of cellular shape descriptors, a method known as cellular morphometry. However, standard C. elegans live imaging methods limit the capability of cellular morphometry in 3D, as spherical aberrations generated by samples and the surrounding medium misalign optical paths. Here, we report a 3D live imaging method for C. elegans embryos that minimized spherical aberrations caused by refractive index (RI) mismatch. We determined the composition of a refractive index matching medium (RIMM) for C. elegans live imaging. The 3D live imaging with the RIMM resulted in a higher signal intensity in the deeper cell layers. We also found that the obtained images improved the 3D cell segmentation quality. Furthermore, our 3D cellular morphometry and 2D cell shape simulation indicated that the germ cell precursor P(4) had exceptionally high cortical tension. Our results demonstrate that the RIMM is a cost-effective solution to improve the 3D cellular morphometry of C. elegans. The application of this method should facilitate understanding of C. elegans morphogenesis from the perspective of cell shape changes.