After preparing agarose with tricaine according to the text protocol on the stage of the stereomicroscope, use a transfer pipette to place four to five anesthetized larvae in a glass bottom dish. Remove the tricaine and agarose solution from the 55-degree Celsius water bath, and let it cool at room temperature for 1 to 2 minutes.
In the meantime, remove as much liquid from the anesthetized larvae as possible. Next, pour the cooled agarose into the dish until about half of the surface is covered. Then, swirl the dish to spread the agarose. Pick up larvae that have floated to the sides of the dish and pipette them back into the center. Then, under the stereomicroscope, use a long pipette tip to gently position the larvae as desired.
For imaging the otic vesicle, position the larvae so that the right otic vesicle is facing up and the left otic vesicle is flat against the bottom of the dish. Let the agarose cool for about 10 minutes before moving the dish. Then, gently pipette some tricaine and E3 solution on top of the agarose layer to keep it moist.
To visualize the samples, use a z-stack scan with 488-nanometer and 543-nanometer lasers. Use continuous line scanning to adjust the laser power and detector gain. Verify that there is no accidentally photoconverted red fluorescence. On the "Image Acquisition Control" window, under "Stimulus Setting," select the "Use Scanner" tool and choose "Main." Select the 405-nanometer laser, and set it to 70% power. Then, using the circle option, define the region of interest in the otic vesicle.
Under the "Stimulus Start setting," select "Activation in Series" with a pre-activation of 1 frame and an activation time of 60,000 milliseconds. On the "Acquisition Setting" window, under the "Time Scan" heading, choose two intervals of minute, each one for pre and one for post-photo conversion. Start the time-lapse series to initiate photoconversion of the defined region of interest. Finally, scan the sample using a z-stack and the 488- and 543-nanometer lasers to visualize the photoconverted red fluorescence as well as any remaining green fluorescence.
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