Probing Cell Mechanics with Bead-Free Optical Tweezers in the <em>Drosophila</em> Embryo

Embryonic development is a highly reproducible process during which the cells and tissues deform to shape the future animal. Such deformations have been shown to require the active generation of forces at the cell level^1,2. To understand morphogenetic processes during which cells and tissues change their shape, it is therefore key to assess the mechanical properties of the cells involved, and to measure the forces within the tissue during the process^3,4. Epithelial layers, especially in Drosophila, have been widely studied due to their quasi-2D geometry and to their easy manipulation.

A number of techniques have thus been developed by us and others to assess epithelial mechanics in vivo during development. We will give a quick overview of the three main techniques used in epithelial tissues. Laser ablation, a widely used method, allows to reveal the local mechanical stress at cell junctions^5,6,7,8 or at larger scale^9,10,11 by performing local cuts that disrupt the mechanical integrity of the target. The dynamics of the opening following the cut provides information both on the stress prior ablation, and on the rheology of the tissue^12,13. A drawback of laser ablation is that it is invasive, as it requires the local disruption of the cell cortex. Hence, one can only perform a limited number of ablations if one wants to preserve tissue integrity. Another drawback is that ablations only provide relative estimates of tension at cell contacts, since the opening velocity is dependent on viscous friction, which is generally not known. Magnetic manipulation has also been developed and used in Drosophila, involving either the use of ferrofluids¹⁴ or ultramagnetic liposomes¹⁵. They can provide absolute measurements^16,17, but are also invasive in the sense that they require the injection of probes at the desired location. This can be very tricky depending on the system, which is not always amenable to precise injections. A third technique, fully non-invasive, is force inference^18,19,20. Force inference relies on the assumption of mechanical equilibrium at triple points (tricellular junctions, or vertices), and allows to infer tensions at all cell-cell contacts (and possibly, pressures in all cells) by solving an inverse problem. For tensions, each vertex provides two equations (X and Y). This yields a large system of linear equations which can be inverted under some conditions to assess tension at all cell contacts. While this method is very attractive, as it only requires a segmented image and no extra experiment or setup, its accuracy is yet to determine, and again it only provides relative values, unless an absolute calibration measurement is performed.

To overcome some of these limitations, we introduce in this article a setup of optical tweezers coupled to a light sheet microscope to apply controlled forces at the cell scale in the embryonic epithelium of Drosophila melanogaster. Optical tweezers have been used for numerous biological applications including the measurements on single proteins and manipulation of organelles and cells²¹. Here, we report applied forces in the range of a few dozen pN, which is small yet sufficient to induce local deformations of cell contacts and perform mechanical measurements in vivo. Typically, we use perpendicular deflection of cell contacts, monitored through the analysis of kymographs, to relate force to deformation. Importantly, our setup does not require the injection of beads at the desired location in the tissue, as optical tweezers are able to directly exert forces on cell-cell contacts. The coupling of the optical tweezers to a light sheet microscope allows one to perform rapid imaging (several frames per second), which is very appreciable for a mechanical analysis at short time scales, and with reduced phototoxicity, since the illumination of the sample is limited to the plane of imaging²².

Overall, optical tweezers are a non-invasive way to apply controlled forces at cell contacts in vivo in the Drosophila embryo, and to extract mechanical information such as stiffness and tension at cell contacts²³, rheological properties²⁴, and gradient or anisotropy of tension²³.