Edward Polanco, Post 1

           The first week during the summer, following finals, when I arrived at the lab, there were 30 new students all eager to begin working on summer research. Since I am one of the more senior members of the lab, I was expected to take on a leadership role helping the new students to begin their training, and to help them find their niche within the lab. I helped get them organized and gave them each jobs to work on that the other senior members in the lab and I thought they could reasonably finish by the end of summer. Often times they would come find me when they have questions, and after hours they would email me if there was any questions they had or if they were unsure of what to do the next day, or if they needed me to point to journals to help them get started, or if they needed clarification on the journals that they were reading. This went on much more during the first two weeks while they were still acclimating to our specific lab environment, but has slowed down considerably since then.

            During last quarter I began working on a line confocal microscope in order to study both the behavior of c. elegans when exposed to a thermal stimulus (an infrared laser) and how their neurons respond to the stimulus as well. We are using an infrared laser for the thermal stimulus because we currently do experiments using a temperature gradient, but we would like to try to create a virtual temperature stimulation so that we can compare the results of the two experiments. Ultimately however, the goal is to learn how the motor neurons and the sensory neurons communicate with each other in order for the worm to be able to be able to tell the difference between whether it runs into an obstacle or whether the obstacle ran into it (like another animal). The former of these events represents only an obstacle whereas the latter could represent a potential threat, therefore being able to distinguish between the two is important. My project is to construct a microscope that integrates the worm tracker developed in our lab, with an infrared laser to stimulate the worm thermally. Currently we use ST2 worms, which are c. elegans with their sensory neuron labeled with GFP, but soon we will have worms with their neurons labeled with GCaMP. This will allow us to study their neurodynamics when exposed to the infrared laser stimulus.

            My first step this summer was to try to get a 3D image of a c. elegan using the epifluorescence microscope I had built during the quarter (the first step towards building the line confocal is to have a working epifluorescence microscope). We did this by paralyzing a worm, and putting it on a stage that can be moved with micron precision. We took 100 frames, each one spaced 2 microns apart, and then reconstructed a 3D image of the worm using Vaa3D (Vaa3D is an open source software package maintained by Janelia Farm). The image came out successful, and we decided to continue with the project. I have since pulled the microscope apart to rebuild it in a more space efficient manner. The current rendition of the microscope is a little unstable because of the long poles which support it, so soon I will purchase a breadboard to remount the microscope onto in order to increase the stability. There are some design nuances which I am trying to work out in order to increase versatility but those will come to fruition as the summer continues. We are also working on optimizing the worm tracking software for this particular microscope because the programming logic is slightly different than it is for the other worm tracking microscope that it was originally developed for. The current status of the worm tracker for this microscope works, but it moves to slow, so we are reworking the programming in order to allow it to modulate the speed appropriately for the speed of the worm. We are also working to modulate the intensity of the infrared laser so that the software can change the virtual heat stimulation that the worm is experiencing.

            The next major improvement to be made is to improve the stability by mounting the microscope on a breadboard, and then to get the laser and the motorized stage working. Once the laser and stage are working, we will need to integrate the software together into one program that controls both. Only then will we be able to redirect our focus on the line confocal and the piezo z-scanning in order to obtain high speed 3D images.

 

First Construction of the microscope. Beam path is closed as much as possible in order to prevent scattering. It will be interesting when the project is finished to see how different it ends up looking.

Taking data for the first time using this microscope. The worm is very small, only about 1mm long, with a diameter of about 100 microns. The blue laser is used to stimulate GFP so we can see the worm’s sensory neuron, AFD.

Preliminary data taken with this microscope. It’s not a particularly good image, but again it will be interesting to see the improvement by the time the project is finished. It is a 3D image that can be rotated, unfortunately that cannot be seen here.

This is the first reconstruction of the microscope. It looks a bit different than the original construction, but is still a far cry from its final form. In this picture the camera is not attached, but it goes in the same place as it was originally.