A Versatile Technique for Measuring the Stiffness of Small Organisms Based on Microscopic Observation of Cantilever Strain

Abstract

Small organisms such as water fleas and nematodes change their body stiffness in response to external stimuli and environmental alterations. Atomic force microscopy (AFM) is commonly used for stiffness measurement. However, the compression of the object by the cantilever is performed in the same direction as the observation, and this makes it difficult to observe the deformation of the object. Furthermore, the placement of the cantilever relative to the object is spatially quite limited and the driving distance is also very short: these limitations complicate measurements in organisms of various shapes and sizes. Here, we developed a new stiffness measurement technique using a cantilever attached to the tip of a micromanipulator, thereby overcoming these limitations of conventional AFM. During the compression of the object vertically downward, we observed the deformation from the side using a microscope tilted at 90°. The cantilever strain was monitored directly under the microscope rather than using the conventional optical reflection method. We used this approach to measure the stiffness of three small animal species, i.e., a tardigrade (Grevenius myrops), a nematode (Caenorhabditis elegans), and a water flea (Daphnia magna). We videotaped the compression of these organisms, and determined the strain distance of the object and cantilever from the images at different time points. A stress-strain curve was analyzed by plotting and Young’s modulus was obtained as the curve’s slope. Thus, Young’s modulus data could be reliably obtained for varied organisms using the same device. This technology will facilitate measurements of stiffness of various small organisms.