Edward Polanco, Post 2

            For the second part of the summer, my PI wanted me to focus less on the construction of the line confocal microscope and more on the science that we plan to do with it. The goal of the line confocal microscope is to be able to stimulate the worms with both light and heat in order to observe avoidance behavior in the worm. In addition to being able to observe behavior, the microscope is also designed to observe neural activity in the worm. In the end we hope to correlate neural activity with behavior in order to identify neurons associated with avoidance behavior, neurons associated with the inhibition of other neurons, and ultimately neurons that might be involved with the central pattern generator.

            Since we wanted to get started on taking data, we built a microscope for studying just the avoidance behavior of the worms. To do this we setup a 2D stage with a joystick in order to move the sample with micron order precision. We used a blue laser focused by an objective lens onto the sample in order to get a small enough spot to hit only the head of the worm. The laser is controlled by Labview in order to be able to turn it on and off with millisecond precision. We used a Cannon 60D camera controlled by Labview for the imaging itself. The setup mostly works, however we are still struggling to get the beam spot small enough to stimulate only the head.

            One of my other recent jobs in the lab has been to work with all of the lasers in the lab. Dr. Arisaka wanted to put one student in charge of all of them who knew where they all were at any given time and how to operate them in case people ran into problems or had questions about them. Since he put me in charge of this the first thing I had to do was measure their power output, since we needed this to be calibrated for any experiment we do with them. One of the interesting things I learned from calibrating the lasers is that most lasers have a stable and predictable range through which the power can be modulated, but as they are modulated to lower powers they become much less stable. I graphed the power outputs using excel in order to identify the range over which the lasers are more stable, and can be modulated in a predictable manner.

            Once I had made excel graphs, Dr. Arisaka wanted me to move on to using Matlab to be able to profile the beam. To do this we needed an image of the beam, and he wanted me to write a Matlab program that uses the image of a beam spot to profile its intensity. I have not finished the program yet because it does not yet incorporate the measured power of the beam nor the field of view of the camera in order to rescale the graphs in terms of intensity and distance. Currently the graph ranges from 0 to 441.6, and the domain is in pixels. The way that the program works is it takes in an image, and stores it as an array. It then goes through each pixel and finds the most intense pixel and records that intensity. It then scans each row and column to find the row and the column with the greatest number of pixels with that intensity. Once it finds the correct row and column, it uses those cross-sections of the image and plots a graph of intensity vs pixel. It also displays the image that it took in, and draws two lines on it, to show which row and column it used to make the graphs.

            The last major interesting thing that I did, was I had to present a journal for the lab about spinning disc confocal microscopy. I had to then compare it to line confocal microscopy because that is what we actually use in our lab. It was fun, I learned a lot from giving the presentation too (I think I learned about as much by giving it, as I did from preparing for it). It was a fun but challenging experience.

            The next steps I need to take are going to be optimizing the microscope for the phototaxis behavior experiment so that the laser stimulates only the head. This will make it so that we have better control over the experiment so we know exactly the intensity, which caused the worm to respond. This is critical to being able to reproduce published data. I then need to finish the Matlab program, so that anytime we use a laser during experiments we only need to measure the power output of the laser and then image a beam spot on the experimental setup and the Matlab program will be able to do all of the heavy lifting to tell us exactly what the intensity profile of the beam is.

Microscope constructed for the phototaxis behavior experiment. Laser comes in from the left and it focused onto the specimen. It then goes upwards to the Basler IR camera at the top. The stage is programmed to keep the worm underneath the objective lens.

Measuring the power output from the laser. The beam exits the laser and is projected onto a blackbody-measuring device. The measurement is then sent to the monitor, which displays the current power, minimum power, maximum power, and average power. It also calculates the standard deviation which determines the stability of the laser.

These are graphs of the power output from each laser. When plotting the graphs, I would start at maximum power, measure the power output for about 2 minutes, and then record the average and modulate the laser down by 10% power.

This is the output from my Matlab program. The program definitely needs work, but so far it is able to find the center of the beam by itself. It then outputs graphs to show the intensity along each row and column (the top graph is a vertical cross section and the bottom graph is a horizontal cross section). It then shows on the original image where the cross sections were taken from in the image.