Blog #2: Path Finding Mechanisms in Transgenic Zebrafish

Throughout the course of the previous 2 months, Dr. Steiner and I have had the opportunity to conduct multiple ablation experiments involving the new Confocal microscope. The assembly of this new microscope that Dr. Steiner was able to get for our research was recently completed, and we were very eager to try it out for the purposes of our 24 hour time lapse microscopy films. All of our previous 24 hour Time lapse microscopy films were conducted on a different Confocal microscope that has been in the lab for quite some time. In the conduction of numerous experimental trials on this microscope, we have never ran into any problems  regarding the imaging procedure, DAPI laser ablations, and 24 hour time lapse that we couldn’t solve. We’ve certainly encountered some challenges, particularly in establishing optimal DAPI laser control for the ablations, but have been able to strategically work through them in experimentation. The adversity we faced early on in the semester has certainly spilled over into our attempts to get the new Confocal microscope up and running as a primary component of our experimental trials.

During our analysis of the 24 hour time lapse films conducted on the new Confocal microscope, we have been consistently confronted with a major problem. All of the 24 hour time lapse films taken on the new Confocal microscope showed that the gaps produced in the interneuromast chains from the laser ablations are not closing. The projections from both sides of the ablated interneuromast chain showed minimal degrees of activity early on in the majority of the films that we reviewed, and failed to find each other and close the gap every single time. This failure of the gap produced in the interneuromast chain to close at this level was something that we never observed when conducting trials on the other Confocal microscope. Dr. Steiner and I are determined to find out why this problem is arising in the 24 hour time lapse films conducted on the new Confocal microscope. Our plan of action is to perform laser ablations on 6 Zebrafish during one morning trial, and image 3 of the Zebrafish on the older Confocal microscope while simultaneously imaging the other 3 on the new Confocal microscope. Review of all 6 of these 24 hour time lapse films should cast light on whether this failed gap closure is emanating from a component of new Confocal microscope or if it has an ulterior origin.

Aside from this problem that we are currently staring into the eyes of, my abilities and competency in carrying out full scale ablations has continued to grow under the direction of Dr. Steiner. Throughout the month of November, Dr. Steiner presented me with multiple opportunities to independently conduct ablation experiments on the Confocal microscope which was very exciting. I had a chance to really test my understanding and ability to apply the things that Dr. Steiner taught me about the microscope during live trials. To me, opportunities of this nature are inherently the times in which real, tremendous growth can happen. In terms of our game plan for the remainder of this month, we plan to solve the problem pertaining to the 24 hour lapse films captured on the new Confocal microscope and to continue to make strides in gathering data points.

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