Misguidance in Path Finding Mechanisms in Twitch-Twice-Roundabout3 Mutant Zebrafish

Over the course of the previous few weeks, Dr. Steiner and I have continued to conduct Laser ablation experiments on the Confocal Microscope. Our primary objective has been to completely ablate the interneuromast chain in an effort to characterize the regenerative process through a 24 hour Time-Lapse Microscopy movie. We have been strategically ablating significant components of the interneuromast chain including cell bodies between neuromasts L3 and L4. In order to appropriately gauge laser power, we developed 3 primary controls based on previous experimental findings that all ascertained a high energy wavelength of 405 nm. In our last experimental trial, we annihilated the interneuromast chain with a laser power of 75% for exactly 60 seconds; and were successful in producing a small-scale gap in the chain. The gap we created confirmed that the control we implemented regarding Laser Power was optimal, and our Time-Lapse movie revealed very interesting results emanating from the ablation itself.

Following a careful reviewing of our 24 hour Time-Lapse Microscopy movie, we were able to clearly identify the gap in the interneuromast chain that we produced and observe its complete regeneration. This particular Time Lapse movie was important not only because it is definitive evidence of the interneuromast chain’s regenerative capacity, but also due to the fact that we observed cell body migration. The cell body migration that we witnessed on the film was slightly odd, and we will remain cognitive of this migration as we progress in experimentation. An understanding of the cell body migration and its function in the regeneration that we observed is something we will be striving for. While this experiment in itself was moderately successful, we will be focusing on creating larger-scale gaps in our continued ablation experiments. Upon successfully ablating the interneuormast to a greater extent, we will maintain the conditions in completing an additional 10-12 experiments to generate statistically significant data.

We have encountered some challenges throughout the course of our experimentation pertaining specifically to the interneuromast chain ablations. We have had some difficultly with actually ablating the interneuromast chain completely, and have noticed that some of our laser power controls only resulted in photobleaching of the chain. Incomplete ablations resulting in mere photobleaching are insufficient, and cannot be used to paint a picture of regeneration. Increasing the laser power to 75% for an additional 60 seconds appears to be a step in the right direction, and is a control that will be built upon. An additional confocal microscope with a spinning disk system is in the process of being assembled, which will function as a definitive solution in later weeks. Additionally, the mating behaviors and patterns of the Zebrafish, our model organism, have presented some adversity as well. In attempting to keep their breeding consistent, they receive multiple feedings on a daily basis.

The past few weeks have been particularly exciting for me, as my degree of competency on the Confocal Microscope is continuing to grow under Dr. Steiner’s mentorship and instruction. I am becoming progressively more comfortable in operating the Microscope during the experimental trials, and will soon be able to carry out experiments independently. The ability to conduct experiments on the Confocal Microscope confidently and soundly is a skill that I consider to be invaluable due to the sheer power of the microscope itself. The immense potential that it brings to the table relative to all walks of scientific exploration is truly incomprehensible. I believe that enhancing my capability and comfortability with the Confocal microscope is something that I can greatly draw upon on in my future in research. I plan to learn absolutely as much as I can about the nature of the microscope, and will take my skills as far they can possibly go. With regards to the future of this experiment, we will continue to work diligently in conducting experimental trials until we have arrived at characterization of interneuromast chain regeneration.

Relevant Questions:

What biological mechanisms are driving the cell body migration that we observed?

How can we continue to adjust the laser power to avoid photobleaching and achieve complete ablation?

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