To begin, turn off the room light. Select Ocular under the ocular panel in the FlowView software. Change the cube turret to four TRITC, and turn off the touch panel controller by clicking off under backlight of touch panel controller in the software.
Then turn off the computer screen. Open the front side of the black cloth cover on the microscope. Take the 35 millimeter glass-bottomed dish containing the embryos from the black box and place it on the microscope stage.
Then turn on the fluorescence illumination unit, and use the eyepiece to identify the embryo of interest. Bring it into focus. Close the black cloth cover to protect the sample from light.
Turn on the computer screen to access the software that controls the microscope. Change the ocular to LSM in the software for image acquisition. Perform laser ablation in control unstimulated embryos using a 25 times magnification water immersion objective.
Click Bright Z, Sequence Manager, and LSM stimulation from the tool window. Set the scanner type as Galvano, and the scan size as 512 by 512. Turn on channel one and channel three under the PMT setting panel to allow the use of the 1040 nanometer laser, and click live four times speed to visualize the embryo.
Rotate the embryo using the rotation function to make the anterior posterior axis vertically oriented, and set the zoom to three. Draw a region of interest or ROI using the shape tool under scan settings, and set the ROI size in the reference panel. Next, set the ROI as 512 pixels in width and 100 pixels in height.
To set the acquisition parameters for the pre-ablation Z stack, register the embryo's surface as zero under the Z section. Set the start as zero and the end as 100 micrometers. Set the step size as two micrometers, and activate the Z acquisition mode by checking Z under the series tab.
Using the bright Z function, set the 1040 nanometer laser intensity to increase linearly from 3%to 7%Save the current imaging setting as the first task of the pipeline by clicking LSM in Sequence Manager. To set acquisition parameters for the pre-ablation movie, set an ROI of 512 by 512 pixels near the embryo's ventral surface as previously demonstrated. Set the 1, 040 nanometer laser intensity to 3%Check time, and uncheck Z under the series panel.
Keep the interval as free run under the time lapse panel, and set the cycle as 10. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. To set the parameters for laser ablation, define a 3D region immediately below the vitelline membrane.
Set the start of the Z stack as the plane, and the end as 20 micrometers deeper. Set the step size as 1.5 micrometers. Turn on channel two and channel four under the PMT setting panel to allow the use of the 920 nanometer laser.
Set the intensity of the laser to 30%and set image acquisition with the laser for a single Z stack within the defined 3D region. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. To set acquisition parameters for the post ablation movie, set image acquisition for a 100 frame single Z plane post ablation movie using the 1, 040 and 920 nanometer lasers.
Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Select sequence under acquire. Change the data saving path and file name as needed.
Click ready, and wait for the software to initialize the pipeline, then click start to execute the pipeline. to perform laser ablation in stimulated embryos, Set acquisition parameters for the pre-ablation Z stack as demonstrated for the unstimulated embryos. Save the current setting as the first task of the pipeline by clicking LSM in Sequence Manager.
To set parameters for optogenetic stimulation within a defined ROI, change the zoom to one, and select an ROI that covers the embryo's ventral surface. Turn off the channel one to channel four detectors, click LSM stimulation, uncheck continuous within duration, and type 12 seconds. Save the current setting as the next task of the pipeline by clicking stimulation in Sequence Manager.
Set a three minute wait time after stimulation to ensure total inactivation of myosin and apical F-actin disassembly and achieve a static tissue morphology before laser ablation. Next, set acquisition parameters for the single Z plane pre-ablation movie as demonstrated for the unstimulated embryos, except that the 1, 040 and 920 nanometer lasers are used for image acquisition. Turn on the channel one to channel four detectors.
Set the intensity of the lasers to 3%and 0.3%Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Set parameters for laser ablation as demonstrated for the unstimulated embryos. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager.
Set acquisition parameters for the single Z plane post-ablation movie as demonstrated for the unstimulated embryos. Save the current setting as the next task of the pipeline by clicking LSM in Sequence Manager. Select sequence under acquire.
Change the data saving path and file name as needed. Click ready and wait for the software to initialize the pipeline, then click start to execute the pipeline. In the unstimulated embryos undergoing apical constriction, spaghetti squash mCherry became enriched at the medial apical region, whereas CRY2Rho one dominant negative mCherry was cytosolic.
In the stimulated embryos, the CRY2Rho one dominant negative mCherry signal became plasma membrane localized, whereas the medial apical signal of spaghetti squash mCherry completely disappeared. Laser ablation of the unstimulated embryos within the constriction domain led to a rapid tissue recoil along the anterior posterior axis, whereas laser ablation in the stimulated embryos did not result in noticeable tissue recoil. The ablation was quantified and shown here.
