Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Our protocol provides both visual and real-time information about how the process of ocular morphogenesis occurs under both normal and pathological conditions. Our protocol allows for the rapid acquisition of z-stacks, which decreases the amount of time between each 3D image in the dataset. This provides an increased temporal resolution of how ocular morphogenesis occurs.

Positioning the embryo within the capillary can be challenging due to its spherical shape at the single somite stage, so it's important to load multiple embryos into the capillary to ensure that at least one is in the correct orientation. To screen for the presence of somites at 10 hours post-fertilization, place the embryos under a stereo microscope and use a fluorescence adapter to confirm the presence of the rx3:GFP transgene. Once three to five GFP-positive single somite embryos have been identified, use fine forceps to decorionate the embryos.

To embed the embryos in agarose, transfer the embryos to a microcentrifuge tube containing 500 microliters of E3 embryo buffer supplemented with 168 micrograms per milliliter of tricaine and add 500 microliters of cooled but liquid 2%low melting temperature agarose and E3 buffer to the tube with gentle mixing. Use a one millimeter glass capillary with a PTFE plunger to pull the agarose-embedded embryos into the capillary and let the agarose solidify for 30 to 60 seconds at room temperature. Then place the capillary into a beaker of tricaine-supplemented E3 buffer.

To set up a lightsheet microscope for embryo imaging, place the capillary into the capillary sample holder and click the metal sheath in the center of the metal disc of the holder. Place two rubber stoppers into the sheath with the slits facing the ends of the sheath and slide the capillary through the middle of the rubber stoppers. Once the marker is at the base of the metal sheath, use the metal cap to secure the capillary in place and place the holder on the top of the microscope with the white marks aligned.

Close the lid of the sample holder and click the locate capillary button in the microscope software interface. Using the Ergo Drive control panel, move the capillary to just above the objective. After opening the lid, gently push the plunger until the section of agarose containing the embryo is hanging below the capillary bottom in front of the objective.

Turn off locate capillary and click locate sample to switch the view from the sample chamber webcam to the microscope objective and use this view to adjust the position of the sample more precisely. Then turn off the locate sample and switch to the acquisition tab. Check the z-stack and time series boxes.

In the acquisition mode parameters window, select the dual side lightsheet setting and check the boxes for online dual side fusion and pivot scanning. Click continuous to obtain a live view of the embryo. In the channel's window, select 488 channel and set the laser power to one and the exposure time to 7.5 milliseconds.

Use the Ergo Drive control panel to adjust the position of the embryo until the eye field directly face the camera and adjust the left and right lightsheets in the channels parameters until the eye field is sufficiently in focus. Set the first and last z-positions around 500 micrometers beyond the last detectable fluorescent signal and click optimal to set the step size to 0.477 micrometers. To set the incubation parameters, check the box for the Peltier unit to keep the temperature at 28 degrees Celsius.

In the time series window, select the appropriate experimental frequency and time interval to acquire the images. Click start experiment. Select the folder to save the image set and set the image prefix, then click save to start the imaging.

For image analysis, import the image files into an appropriate 4D image analysis software program and click 4D viewer cube, scale bar, and the video icon to open the storyboard taskbar. Select add keyframe sequence and specify the duration of the video in seconds. Uncheck the create rotation box and check use time progression to include specific time points in the video.

Save the storyboard to apply the same parameters to multiple image sets. Click export movie to save a video of the timelapse imaging and specify the movie export settings, including the filename and location, video format, video resolution, frame rate, data resolution. Add timestamps if desired and select record.

To create rotation videos at specific time points, add a keyframe sequence as just demonstrated, but check the create rotation box and uncheck the use time progression box. To render a high resolution image at any orientation and at any individual time point, select the camera icon in the 4D viewer. To build a pipeline for analysis, select flask to access the analysis panel and open the analysis operations menu to select the sequence of operations of interest.

Click the blue triangle to initiate the pipeline. At the end of the run, click feature columns in the popup window to view a list of the features that can provide information about the object of interest, then click export to export the data to a spreadsheet. In this representative analysis, a transgenic rx3:GFP embryo was imaged from the dorsal vantage point every five minutes from the one somite stage through 24 hours post-fertilization for a total of 14 hours.

As observed, performing a pipeline run of the images captured from this analysis facilitated the generation of a mask of the developing eye. Note that when the eye field is separated into two optic vesicles, a third rx3:GFP positive region in the forebrain that contributed to the hypothalamus could be observed. This information was also observed in the volume data and can be easily separated from the optic vesicle data as it was much smaller in volume than that observed for either optic vesicle.

This procedure can be followed up with additional analyses, for example, tracking individual cells across the image dataset.