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
