Force microscopy of the Caenorhabditis elegans embryonic eggshell

A tough egg to crack By carefully probing the surface of Caenorhabiditis elegans eggs, researchers were able to quantify the physical strength of the various layers shielding the developing embryo. This worm species is a popular laboratory model, and the general structure of its embryonic eggshell has been closely analyzed. Little is known about the shell's mechanical characteristics, however, and researchers led by Roger Krenger of the EPFL and Jan Burri of the ETH in Switzerland employed a technique called cellular force microscopy (CFM) to address this question. CFM entails precisely prodding biological specimens with a microscale probe and collecting measurements of the resulting force. This allowed them to not only measure the rigidity of the intact egg, but also to assess the structural contributions of individual shell layers by performing measurements after using chemical treatments to remove these various strata. Assays focusing on emerging biological phenomena in an animal's life can be performed during embryogenesis. While the embryo of Caenorhabditis elegans has been extensively studied, its biomechanical properties are largely unknown. Here, we demonstrate that cellular force microscopy (CFM), a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy, can be successfully applied to C. elegans embryos. We performed, for the first time, a quantitative study of the mechanical properties of the eggshell of living C. elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments. In addition to investigating natural eggshells, we applied different eggshell treatments, i.e., exposure to sodium hypochlorite and chitinase solutions, respectively, that selectively modified the multilayer eggshell structure, in order to evaluate the impact of the different layers on the mechanical integrity of the embryo. Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves. We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell. Furthermore, our results showed, in contrast to previous assumptions, that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell, but also significantly degenerate the underlying chitin layer, which is primarily responsible for the mechanical stability of the egg.