Live Cell Imaging of Muller Glial Nuclear Migration During Zebrafish Retinal Regeneration

Begin with an agarose-embedded dorsal retinal explant from a transgenic zebrafish, mounted in a fluorodish with media.

The retina exhibits light-induced photoreceptor damage and contains Muller glial cells or MGCs with nuclei expressing GFP.

Place the dish into a multiphoton microscope chamber.

Using bright-field illumination, focus on the retina.

Switch to fluorescence mode to locate the MGCs.

Configure a z-stack from the ganglion cell layer or GCL to the outer nuclear layer or ONL.

Start live imaging.

Damaged photoreceptors release TNF-α, activating the MGCs.

MGC nuclei migrate basally within the inner nuclear layer or INL to replicate their DNA.

Then, the nuclei migrate apically to the ONL to undergo mitosis, producing neuronal progenitors.

After mitosis, the MGC nuclei and progenitors migrate basally back to the INL.

The progenitors proliferate, migrate to the injury site, and differentiate into photoreceptors, enabling retinal regeneration.

Analyze the captured images to visualize nuclear migration.

In the image acquisition software, open A1 MP GUI, TiPad, A1 Compact GUI, and ND Acquisition windows. For multiphoton imaging, ensure that the IR NDD Option is chosen in A1 Compact GUI window. Then, in the setting field, select IR-DM for the first dichroic mirror and check that the bandpass filter is set to 525 to 50 to acquire GFP fluorescence.

Next, switch on the IR laser in the A1 MP GUI window. It will take a few minutes for the laser to be ready. After that, set the wavelength to 910 nanometers to excite GFP fluorescence and align the laser by clicking the Auto Alignment button.

After placing the retinal culture on the multiphoton microscope stage, ensure that the room and equipment lights are switched off before opening the shutter to avoid overexposure of the photomultiplier tube. To reduce noise levels, house the microscope in a darkened environment.

Acquire images of a field of view of 300 by 300 pixels at a zoom of 2, which are set in the Scan Area window, and a pixel dwelling time of 4.8 microseconds per pixel chosen in the A1 Compact GUI window. Roughly set up the laser power by changing the acquisition area in the A1 MP GUI window and the gain in the A1 Compact GUI window.

Now, focus on the ganglion cell layer and set the top focal plane of the z-stack in the Z sub-window within the ND Acquisition window. Move the focal plane through the level of the outer nuclear layer, which is characterized by the presence of dimly labeled GFAP and GFP-positive cells that are round and enlarged relative to their counterparts in the inner nuclear layer.

Set this plane as the bottom of the z-stack. Next, set the z step size between 0.7 to 1 micrometer. To set up the z intensity correction, open the Z Intensity Correction window and choose From ND for setting the z-stack range. Then, click on the bottom focal plane in the Z Intensity Correction window and set the laser intensity and gain.

Afterward, click the arrow next to the z values in the Z Intensity Correction window to confirm the settings that are subsequently shown under Device Settings in the Z Intensity Correction window for the chosen focal plane. Repeat the process for the middle and top planes, increasing the laser power and gain.

Set the acquisition area in the A1 MP GUI window and avoid selecting an acquisition area larger than 15 and a gain higher than 126 at the start of imaging to circumvent photobleaching and increased noise levels. Subsequently, choose relative intensity correction in the Z Intensity Correction window. In the time series sub window of the ND Acquisition window, set the duration to eight hours and the interval to no delay. Then, click the Run Z Correction in the Z-Stack sub-window of the ND Acquisition window to acquire 3D time series.