Over the course of the previous two months, Dr Steiner and I have continued to push forward in our laser ablation experiments. The transgenic Zebrafish that our studies revolve around have been breeding consistently every week, which has been excellent and enabled us to continue to conduct experimental trials. We have maintained the same controls pertaining to the DAPI laser, which are a laser power of 75% and wavelength of 405 nm (high energy), but have made some specific changes in the actual ablation. During our imaging on the Confocal microscope, Dr. Steiner began to notice some rather discrete cells that were very dim in terms of fluorescence but clearly visible. These cells were located very close to the site of ablation on the interneuromast chain, and have the potential to completely confound our ablations. We want to be absolutely definitive regarding the fact that we have successfully ablated the interneuromast chain, which entails that all of the cells at that site have been killed. In an effort to prevent these cells from surviving the ablation and confounding our results, Dr. Steiner and I have been zeroing in on them during our experimental trials.
We first began with maintaining the aforementioned laser controls and annihilating these background cells for 10-15 seconds, which was followed by careful interpretation of the subsequent images. We wanted to be sure that we have successfully ablated those cells, and that they would be out of the picture. These additional preliminary 10-15 second ablations enabled us to eliminate these target cells, but have had some blowback on our experimental trials. In the experimental trials where we hit the background cells for 10-15 seconds, we frequently observed that the interneuromast chain failed to close the gap in the 24 hour Time Lapse Microscopy footage. Dr. Steiner and I both feel that the failure of the interneuromast chains to close may have been due to excessive ablation emanating from the additional targeting of these background cells.
These background cells have become a rather challenging dynamic, but we were still able to obtain an additional 2 data points over the course of the last two months. Both of these data points were fairly strong, and made good contributions to our data. Dr. Steiner and I plan to continue to conduct ablation experiments on a weekly basis, but will be setting our sights on using fish that are slightly older. We have been conducting experiments on fish that are 2 days post fertilization, and will be conducting experiments on fish that are 3 days post fertilization in the coming weeks. We are excited to see if there is any difference in our results of experimental trials centered around fish that are slightly older.