Expression of fluorescent reporters in transgenic animals makes it possible to follow cellular responses to experimental manipulations in living disease models. Here, selective cell loss is pharmacologically induced in zebra fish, high resolution multicolor confocal. Images of the targeted cells and immediate neighbors are acquired prior to immediately after, and its spaced intervals following Drug treatment image analysis demonstrates the activation of endogenous stem cell populations and subsequent replacement of targeted cells during the recovery phase.
This method can help answer key questions in regenerate biology field, such as What are the cellular and molecular mechanisms used to regenerate discreet cell types Begin by transferring transgenic eggs collected from matings to a Petri dish containing 0.3 x danio solution at 16 hours post fertilization. ADD PTU to inhibit tyrosine ACE activity prior to any evidence of pigmentation in the tissue of interest. If the embryo is not albino, incubate the embryos at 28.5 degrees Celsius.
Once reporters are evident, place the Petri dish under a fluorescent stereo zoom microscope and sort for desired transgene expression. Prior to imaging. Prepare a 0.5%low melt aeros mounting solution in embryo medium and store it at 40 degrees Celsius.
Add trica and PTU if necessary. Mix gently and return to 40 degrees Celsius. Anesthetize the fish by placing them in embryo medium containing trica.
For approximately three minutes, the fish will become non-responsive. To touch using a micro pipette, draw individual fish into a trimmed pipette tip in a volume of approximately 30 microliters of anesthetic solution. Next, invert the pipette and allow the fish to settle to the bottom.
Touch the tip to the mounting solution and allow them to transfer by gravity. Take care not to dilute the agro so that it can be reused. Discard the remaining anesthetic solution from the micro pipette.
Then using the same tip, transfer the fish to a Petri dish. In about 30 microliters of mounting solution, gently orient the fish such that the region of interest is centered in the aeros droplet. Make sure the fish remain in a desired orientation until the aeros solidifies.
Multiple fish can be mounted in a single Petri dish if desired. After the aeros has solidified, transport the fish to the confocal microscope stage and gently add embryo medium containing trica plus or minus PTU to the dish until the fish are completely submerged. Open the imaging software and focus on the region of interest for optimal imaging of cellular and or molecular detail.
Use long working distance objectives with high numerical apertures in the software. Choose the appropriate flora fours from the dial list. Click on spectral setting and make adjustments to the emission ranges.
To obtain clean separation of reporter signals and to maximize signal to noise ratios. Select the appropriate objective in the dropdown menu to ensure that any software defined presets are adjusted properly. To focus the lasers and set power levels, select an output from the lookup table that reveals pixel saturation for each channel.
Next, set detector sensitivity to obtain the desired image quality, gradually raise the laser output until the image intensity is acceptable. Avoid pixel saturation in the region of interest and keep laser levels as low as possible. To reduce photobleaching and phototoxicity issues.
Ensure that each laser line produces detectable signal only in the appropriate channel by manually turning lasers on and off while monitoring all imaging channels. If no crosstalk is evident, all channels can be acquired simultaneously. Next, frame the area of interest using the zoom and rotation functions.
Then set the scan speed and image size using faster scan speeds, minimizes laser dwell time and increases temporal resolution. Set the Z dimension step size according to experimental needs. Here a step size of 10 microns is used to image seven optical sections of retinal tissue.
Once the appropriate settings have been determined, acquire Zack images and save. Then move to the next fish. Serial catch and release imaging can be performed to track cellular changes over successive days according to the instructions in the accompanying written document.
When performing a multi reporter imaging experiment, it's best to use floor floors that are well separated, spectral, however, that is not always practical. Here we show a simple image subtraction method using the open source software image J to separate floor floors with overlapping excitation and emission profiles. In our case, GFP and YFP.
The timelines imaging techniques described in this video have been used to monitor GFP labeled retinal stem cells during the ablation and regeneration of retinal bipolar neurons To perform spectral separation, collect images using sequential imaging modes and variable barrier filter settings, which allow overlapping floor fours to be acquired independently and partially separated respectively. Once the images have been acquired, use the ImageJ loci bio formas import to open the image files to start the import tool. Drag the file of interest into the image J window.
Split the channels into individual stacks using the split channels checkbox in the import window to start the align three TP plugin. Click on plugins, then align stacks. Then align three tp.
The stacks must be aligned to ensure proper subtraction. Select the reference stack in the first dropdown box and the misaligned stack in the second. Check the use XY relative origin and use Z relative origin boxes and click okay.
To start the alignment process, click the volume registration button. A new window will appear that contains the alignment parameters. Click okay.
This section has been optimized by the distributor of the plugin, so no modifications are necessary. When finished, an aligned stacks window will appear. Select output from the aligned window to open the output window.
This window is also optimized and needs no modification. Click okay. A new stack will appear in the workspace with aligned appended to the end of the file Title.
To remove crosstalk using image calculator, click on process then image calculator. Select the stack to be subtracted in the image one dropdown box and the stack to be used in the subtraction. In the image two dropdown box, select subtract in the operation dropdown box and click okay.
Image J will ask to process the stack. Select yes. A new stack should appear, which has removed the crosstalk between overlapping channels.
Merge the stacks to recreate the same image structure before alignment and subtraction. By selecting merged channels in the channel tools window, the tool allows the merger of up to four stacks into a hyper stack. Finally, colorize the channels adjust for brightness and contrast and save the files as tiffs to examine targeted loss and regeneration of nitro reductase.
M cherry expressing retinal bipolar cells. A time series of confocal images were taken in individual zebrafish prior to treatment. There is mosaic expression of membrane tagged YFP reporter in controlled bipolar cells shown in yellow and nitro reductase cherry fusion protein in targeted bipolars shown in red.
The membrane tagged CFP transgene shown in cyan provides contextual information about most other retinal cells for added clarity. The same image without the CFP signal is shown following treatment with metronidazole. The red nitro reductase expressing bipolar cells are lost while the yellow controlled bipolar cells are spared.
This demonstrates the highly specific nature of this ablation methodology. Again, the same image without CFP is shown for clarity. When metro NIAZ is removed, NTR expressing cells return here.
An example of the G-F-P-Y-F-P subtraction method is shown in this figure GFP labeled Mueller. GL cells are shown in green and YFP labeled bipolar cells are shown in purple. Overlap between the two produces white to perform the subtraction.
The GFP and YFP images must first be aligned. The subtraction process then allows YFP labeled bipolar cells to be removed from the GFP image, allowing GFP labeled Mueller ggl to be cleanly represented and dimmer Mueller cells to be detected. The overlap between dim Mueller, ggl and M cherry labeled bipolar cells shown in red can now be verified.
The data shown here suggests that bipolar cell ablation triggers mullar ggl to replace the lost cells. This interpretation is in keeping with recent studies of retinal regeneration, which implicate Mueller Ggl as injury induced progenitors in both mammals and fish. This technique allow researchers to explore mechanisms regulating the regeneration of specific cell types through visualization of stem cells in zebrafish.
