Abstract
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 P4 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.
Highlights
Live imaging of Caenorhabditis elegans (C. elegans) embryos plays a paramount role in dissecting questions pertaining to both cell and developmental biology
We determined the composition of an refractive index matching medium (RIMM) for C. elegans embryos and adult heads
The RI value for adult heads, which was determined by the previous tomographic phase microscopy analysis (RI: 1.36–1.38; [25]) and the 3D refractive index microscopy analysis (RI: ~1.35; [5]), were slightly different compared to the value obtained by us (RI: 1.379)
Summary
Live imaging of Caenorhabditis elegans (C. elegans) embryos plays a paramount role in dissecting questions pertaining to both cell and developmental biology. Widely used C. elegans live imaging methods with conventional confocal microscopes fails to capture entire embryos and embryonic cell sets at high resolution presumably due to spherical aberrations. The use of non-conventional microscopy systems such as dual-view plane illumination microscopy [6] and lattice light-sheet microscopy [7] allow rapid and high-resolution volumetric imaging. To analyze the RI mismatch between C. elegans samples and the surrounding medium, we imaged embryos and adult heads in different iodixanol concentrations using Nomarski differential interference contrast microscopy (DIC), a common transmitted light microscopy method used by worm researchers. These models were used to perform 3D cellular morphometry of individual embryonic blastomeres
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