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.

1. Setting-up the Light Sheet Microscope
<ol>
	<li>Refer to the description of the setup in previous publication25. 
	NOTE: The setup is composed of an upright microscope stage and a light sheet module producing a light sheet in the horizontal plane. A 10X excitation objective lens directs the light sheet into a glass cuvette (Figure 4). The detection objective lens has a high NA (1.1), which is necessary for efficient tweezing (see below).</li>
</ol>
<p class=...

<ol>
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K., Hufnagel, L., Shraiman, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stress+inference+for+two+dimensional+cell+arrays.">Mechanical stress inference for two dimensional cell arrays.</a> PLoS computational biology. 8 (5), e1002512 (2012).</li><li>Ishihara, S., Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian+inference+of+force+dynamics+during+morphogenesis.">Bayesian inference of force dynamics during morphogenesis.</a> Journal of theoretical biology. 313, 201-211 (2012).</li><li>Brodland, G. W., Veldhuis, J. H., Kim, S., Perrone, M., Mashburn, D., Hutson, M. 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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 border="0" cellpadding="0" cellspacing="0" width="1377">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Ytterbium Fiber Laser LP, 10 W, CW</td>
			<td style="width:179px;">IPG Laser</td>
			<td style="width:179px;">YLM-10-LP-SC</td>
			<td style="width:591px;">including collimator LP : beam D=1.6 mm and red guide laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">&#216;1/2&#34; Optical Beam Shutter</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">SH05</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Small Beam Diameter Galvanometer Systems</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">GVS001</td>
			<td style="width:591px;">1 for X displacement, 1 for Y displacement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">1D or 2D Galvo System Linear Power Supply</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">GPS011</td>
			<td style="width:591px;">galvanometers power supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">2 lenses f = 30mm</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">LB1757-B</td>
			<td style="width:591px;">relay telescope between 2 galva</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Lens f = 200mm</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">LB1945-B</td>
			<td style="width:591px;">2.5X telescope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Lens f = 500mm</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">LB1869-B</td>
			<td style="width:591px;">2.5X telescope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:428px;">Right-Angle Kinematic Elliptical Mirror Mount with Tapped Cage Rod Holes</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">KCB1E</td>
			<td style="width:591px;">Periscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:428px;">Laser Safety Glasses, Light Green Lenses, 59% Visible Light Transmission, Universal Style</td>
			<td style="width:179px;">Thorlabs</td>
			<td style="width:179px;">LG1</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">45&#176; AOI, 50.0mm Diameter, Hot Mirror</td>
			<td style="width:179px;">Edmund Optics</td>
			<td style="width:179px;">#64-470</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Multiphoton-Emitter HC 750/S</td>
			<td style="width:179px;">AHF</td>
			<td style="width:179px;">HC 750/SP</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">CompactDAQ Chassis</td>
			<td style="width:179px;">National Instruments</td>
			<td style="width:179px;">cDAQ-9178</td>
			<td style="width:591px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">C Series Voltage Output Module</td>
			<td style="width:179px;">National Instruments</td>
			<td style="width:179px;">NI-9263</td>
			<td style="width:591px;">Analog output module</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">C Series Voltage Input Module</td>
			<td style="width:179px;">National Instruments</td>
			<td style="width:179px;">NI-9215</td>
			<td style="width:591px;">Analog input module</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoSpheres Carboxylate-Modified Microspheres, 0.5 &#181;m, red fluorescent (580/605), 2% solids</td>
			<td>ThermoFisher Scientific</td>
			<td>F8812</td>
			<td>calibration beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C++ (Qt) home made optical tweezers software</td>
			<td></td>
			<td></td>
			<td>developed by Olivier Blanc and Claire Chard&#232;s. Alternative solution: labview</td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo at JoVE.com

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

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.

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 ...

This method can help answer key questions in tissue mechanics such as how properties of cell cell contacts change in space and time during tissue morphogenesis. The main advantage of this technique is that it is weakly invasive and it is compatible with live imaging such as light sheet microscopy. This technique doesn't require injection of beads in to the tissue.Usually used as demiere probes on which to get froses, I'm excited. Demonstrating the process will be Chardes, an engineer, and Raphael Clement, research scientist from my level of terrain. To begin, set up an upright microscope for light sheet microscopy in the horizontal plain, then prepare the laser and optics to act as the optical tweezers.Next, pipe out one micro-liter of 500 nanometer fluorescent beads in to a glass cuvette, and add 10 milliliters of distilled water. Place the cuvette in the cuvette holder under the objective, and adjust the focus of the objective so that the objective touches the water's surface. Now, open the Integrated Development Environment QT Creator which is used to control the acquisition software.Open the acquisition software, by opening the microRheo. profile, and turn on the live acquisition mode, by selecting live. Prior to switching on the laser, put on safety goggles, then, adjust the laser power to one watt, and switch on the laser.The live feed should now show a bead trapped at the laser's focus. If necessary, adjust the position of the second telescope lens, to observe the bead in focus. One may also need to adjust the angle of the periscope maneuvers to center the bead in the image.Prepare the data acquisition board and the curb connections, as described in figure three of the accompanying text protocol. On the optical shutter interface, adjust the time open sec parameter, to 000.000, the time closed sec parameter to 000.000, the mode parameter to single, and the trigger parameter to EXT. Once set, select the enable button.In QT Creator, open the ot. profile to open the project. Change the name of the input and output in aogenerator.cpp file to match the MI cards that were used in your set up. Finally, compile and run the optical tweezers software. In the acquisition software, select the desired exposure time, the game, the time between two images, the number of images to be acquired, and the laser's power.Then, in the optical tweezers software, set the optical tweezers parameter, shown here, to trace a circle. Next, check the AI parameters box, the weight forward box, and press the record push button, then start the image acquisition. Switch on the optical tweezers, be selecting the generating button, and allow the trapped bead to complete at least two full circles, then stop the optical tweezers, by unselecting the generating button.Finally, stop the image acquisition. Note that a tiff movie, and a text file with galvanometer voltages are created in the default folder. Open Matlab, go to the calibration folder, and run the position two tension script The script computes the interpolation function, translating galvanometer voltages to optical trap position.Next, follow along in the accompanying text protocol to set up the calibration movie. Check if the interpolation map for X and Y laser positions are computed and displayed properly by the script. Check for white regions in the map, which represent missing values.If there are significant white areas, repeat the operation with a new calibration movie. The calibration movie shown here represents a movie with adequate data. Using a pike, or a moist brush, select about 10 embryos at the desired stage and align them.Then use a diamond marking pen to cut a piece of the glass slide, to 10 by 20 by one milometer. Add glue on one side of the cut piece, and let it dry for 20 seconds, then turn the cut piece over, and place it on the line of embryos, sticking it at the edge of the slide. Next, install the preparation in the sample holder, and place the holder in to the cuvette, then place the cuvette on the piso-electric state, and fill it with water.To begin, find the location of interest, and move the target junction's midpoint to the laser trap's position, using the pisar stage. Set the trap's parameters to achieve oscillations perpendicular to the contact line. Also set the phases as zero, the amplitudes at X and Y directions to have a movement perpendicular to the contact line, and set the amplitude to 0.1 volt.Now, start the acquisition, using a fast frame rate, such as 10 frames per second. Once the imaging has begun, switch on the optical tweezers by pressing the go push button. When the imaging is complete, switch off the tweezers, and stop image acquisition.In the specified folder, there will be a movie, and a text file containing the galvanometer voltages during the acquisition. Open the movie to observe the collected frames. Following analysis of the video, measure the stiffness and tension in the tissue sample, by opening the video in Matlab.Here, use the plot function, to plot the interface position as function of the trap position. Then, use Matlab easy fit free toolbox to perform a linear fit of the data. The inverse of the fit's slope, provides the average ratio of the trap position over the interface position.From this information, use the equations in the accompanying text protocol to determine the stiffness, and tension values. The laser trap produces a sinusoidal deflection of the sample, that is perpendicular to the interface. shown here as three successive interface positions.This can also be recorded as movies to generate a climograph and are further analyzed to determine the position of the interface with sub-pixel resolution. Here, the period was five seconds. In the regime of small deformations, the trap and interface positions are proportional.The amplitude of the interface deflection relative to that of the trap gives access to the interface tension or stiffness. Shown here are the basics of a pull and release experiment. When the optical trap is switched on, approximately one micrometer away from the midpoint of the interface between two cells, the interface deflects towards the trap's position.The trap is then switched off after the desired amount of time. The resulting graph can be compared to hypothesized real logical models, such as a Maxwell like model. DOn't forget that working with infrared laser can be extremely hazardous, and precautions such as wearing protective goggles, should have always been taken when performing this procedure.Here, with the most frequent user from tinkle tweezers for those of the embryo, the method can also be used for other mother systems such as the

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

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JoVE Journal

Developmental Biology

7.4K vistas.  Aix-Marseille Université. Este método puede ayudar a responder preguntas clave en la mecánica de tejido, como cómo las propiedades de los contactos celulares cambian en el espacio y el tiempo durante la morfogénesis tisular. La principal ventaja de esta técnica es que es débilmente invasiva y es compatible con imágenes en vivo como la microscopía de láminas de luz. Esta técnica no requiere la inyección de perlas en el tejido.Usualmente se utilizan como sondas demiere en las que conseguir asos, estoy ...