多光子顕微鏡によるライブセルイメージングのための大人のトランスジェニックゼブラフィッシュ網膜外植片の文化

The overall goal of this isolation procedure is to enable live cell imaging of the proliferating cells that are undergoing interkinetic nuclear migration in the retinal explant cultures from damaged zebrafish. This method can answer key questions in retinal regeneration, such as what mechanisms drive interkinetic nuclear migration or whether Muller glia in a renal progenitor cell interkinetic nuclear migration are similarly regulated. The main advantage of this technique is that the dynamic behavior of the proliferating cells undergoing interkinetic nuclear migration during regeneration can be monitored, which permits the investigation of cell cycle stage specific mechanisms.

Though this method can provide insight into interkinetic nuclear migration, it can also be applied to other retinal research areas, such as investigating microglial behavior or Muller glia phagocytosis of dying neurons. Begin this procedure by transferring the zebrafish onto a dry paper towel. Then, remove one of the eyes with a pair of curved forceps and transfer it onto a FluoroDish.

Under a stereo microscope, orient the eye with the pupil facing the coverslip of the FluoroDish so that the optic nerve on the back of the eye is visible. Then, remove the optic nerve as close to the back of the eye as possible. In addition, remove the connective tissue outside of the eye.

Hold the eye between its nasal and temporal sides with a pair of forceps while making an incision at the optic stock by piercing through the lamina cribrosa with one scissor blade and cutting along both the nasal and temporal sides of the eye. Next, pull the dorsal and the ventral sides of the retina apart. Remove the sclera from the dorsal retina with one pair of forceps while holding the lens that is connected to the retina with a second pair of forceps.

Following that, remove the lens and the vitreous without damaging the retina. Then, flatten the retina with the ganglion cell layer facing the coverslip of the FluoroDish. Afterward, surround the retina with 10 microliters of 1%low melting point agarose.

Make sure that the retina is not lifted by the liquid agarose because even slight elevation might affect the ability to focus through the tissue. If lifting is observed, use a pipette to remove the agarose. It is critical that the agarose does not lift the tissue from the coverslip, as this reduces the ability to obtain quality images.

Repeat this procedure several times before adding 1%low melting point agarose to cover the entire FluoroDish. Once the agarose has solidified, add 1.5 milliliters of culture medium to the tissue. In the image acquisition software, open A1 MP GUI TI pad, A1 compact GUI, and ND acquisition windows.

For multi photon imaging, ensure that the IRNDD option is chosen in A1 compact GUI window. Then, in the setting field, select IRDM for the first dichroic mirror and check that the band pass 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 flourescence 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 photo multiplier 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 two, 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 one 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 photo bleaching 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 8 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.

In this procedure, crop a region that contains a dividing GFAP and GFP positive nucleus. Choose a cropped region that contains at least one nucleus that does not undergo INM in order to set a reference point to subsequently measure the distances that the dividing nucleus migrated in relation to the basal INL. It helps to prepare a 3D reconstruction.

Using the show slices view function, generate orthogonal projections. Subsequently, change the mode from slice to maximum intensity projection. Then, turn off the XY view of the orthogonal maximum projection.

Depending on the orientation at which the migrating and dividing nucleus is best visible, also turn off either the XZ or the YZ view. Click on the remaining image with the right mouse button and extract the XZ or YZ image series with the create new document from this view function. If necessary, rotate the image.

Using the manual measurement function, draw a horizontal line across the image at the bottom level of the nucleus that remains in the basal INL and does not undergo INM. Measure the distance between the reference line and the basal point of the migrating nucleus for the time series using the line measurement tool in the analysis software. Shown here is the maximum YZ projection of a z stack image series of the retinal explants at different time points of the time lapse recording acquired by multiphoton microscopy.

Using the line tool under the manual measurement function, a horizontal line was placed at the level of the reference cell indicated by a star. A vertical measurement line was positioned starting at the horizontal line and extending to the basal position of the migrating GF AP and GFP positive nucleus. Distance that the cell, F0, measured in the previous figure migrated apically and that the arising daughter cells, D1 and D2, migrated basally in the multi photon time lapse recording.

The red line indicates the measurements for which the apical migration velocity was calculated, while the black and gray lines indicate the measurements used to calculate the basal migration velocities for D1 and D2 respectively. The distance was plotted against time in itself for apical migration before the nuclear envelope breakdown and the phase of rapid basal migration for daughter cell D1.Once mastered, the isolation of retinas from three fish can be done in 60 to 90 minutes if it is performed properly. This time excludes the preparation steps.

While attempting this procedure, it's important to remember to work as quickly as possible to ensure viability of the retinal explant. It is also critical to mount retinas as flat as possible to enable imaging in deeper tissue layers. After watching this video, you should have a good understanding of how to isolate and mount zebrafish retinas, perform live cell imaging in order to monitor interkinetic nuclear migration and to determine migration velocities.