Name: Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo (Video) | JoVE Journal
Uploaded: 2018-11-02T14:00:00.000+00:00
Duration: 8 min 23 s
Description: Learn bead-free optical tweezers for measuring cell mechanics in Drosophila embryos using light sheet microscopy. This minimally invasive technique enables real-time tension measurements during morphogenesis.

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This work was supported by an FRM Equipe Grant FRM DEQ20130326509, Agence Nationale de la Recherche ANR-Blanc Grant, Morfor ANR-11-BSV5-0008 (to P.-F.L.). We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INBS-04-01, &#171;Investments for the future&#187;). We thank Brice Detailleur and Claude Moretti from the PICSL-FBI infrastructure for technical assistance.

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The authors have nothing to disclose.

Optical tweezers allow to perform absolute mechanical measurements directly in the developing embryonic epithelium in a non-invasive manner. In that sense, it presents advantages over other methods such as laser ablation, which are invasive and provide relative measurements, magnetic forces, which require injections, or force inference, which relies on strong assumptions and also provide relative measurements.
The protocol includes a few critical steps. First, as the objective lens may show ch...

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 level1,2. 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 process3,4. 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 junctions5,6,7,8 or at larger scale9,10,11 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 tissue12,13. 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 measurements16,17, 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 inference18,19,20. 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.

<table><tbody><tr><td>Ytterbium Fiber Laser LP, 10 W, CW</td><td>IPG Laser</td><td>YLM-10-LP-SC</td><td>including collimator LP: beam D = 1.6 mm and red guide laser</td></tr><tr><td>&amp;Oslash;1/2&quot; Optical Beam Shutter</td><td>Thorlabs</td><td>SH05</td><td></td></tr><tr><td>Small Beam Diameter Galvanometer Systems</td><td>Thorlabs</td><td>GVS001</td><td>1 for X displacement, 1 for Y displacement</td></tr><tr><td>1D or 2D Galvo System Linear Power Supply</td><td>Thorlabs</td><td>GPS011</td><td>galvanometers power supply</td></tr><tr><td>2 lenses f = 30 mm</td><td>Thorlabs</td><td>LB1757-B</td><td>relay telescope between 2 galva</td></tr><tr><td>Lens f = 200 mm</td><td>Thorlabs</td><td>LB1945-B</td><td>2.5X telescope</td></tr><tr><td>Lens f = 500 mm</td><td>Thorlabs</td><td>LB1869-B</td><td>2.5X telescope</td></tr><tr><td>Right-Angle Kinematic Elliptical Mirror Mount with Tapped Cage Rod Holes</td><td>Thorlabs</td><td>KCB1E</td><td>Periscope</td></tr><tr><td>Laser Safety Glasses, Light Green Lenses, 59% Visible Light Transmission, Universal Style</td><td>Thorlabs</td><td>LG1</td><td></td></tr><tr><td>45&amp;deg; AOI, 50.0 mm Diameter, Hot Mirror</td><td>Edmund Optics</td><td>#64-470</td><td></td></tr><tr><td>Multiphoton-Emitter HC 750/S</td><td>AHF</td><td>HC 750/SP</td><td></td></tr><tr><td>CompactDAQ Chassis</td><td>National Instruments</td><td>cDAQ-9178</td><td></td></tr><tr><td>C Series Voltage Output Module</td><td>National Instruments</td><td>NI-9263</td><td>Analog output module</td></tr><tr><td>C Series Voltage Input Module</td><td>National Instruments</td><td>NI-9215</td><td>Analog input module</td></tr><tr><td>FluoSpheres Carboxylate-Modified Microspheres, 0.5 &amp;micro;m, red fluorescent (580/605), 2% solids</td><td>ThermoFisher Scientific</td><td>F8812</td><td>calibration beads</td></tr><tr><td>C++ (Qt) home made optical tweezers software</td><td></td><td></td><td>developed by Olivier Blanc and Claire Chard&amp;egrave;s. Alternative solution: labview</td></tr></tbody></table>

probing cell mechanics with bead-free optical tweezers in the drosophila embryo

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Figure 5 shows experimental data obtained by imposing a sinusoidal movement to the trap. The trap produces a deflection of the interface, as exemplified by the 3 snapshots showing 3 successive interface positions (Figure 5A)23. Recorded movies are used to generate a kymograph (Figure 5B) and are further analyzed to determine the position of the interface with subpixel resoluti...

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

probing-cell-mechanics-with-bead-free-optical-tweezers-drosophila

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Learn bead-free optical tweezers for measuring cell mechanics in Drosophila embryos using light sheet microscopy. This minimally invasive technique enables real-time tension measurements during morphogenesis.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

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