It is sad to say that this will be the last post for the academic year; however I have enjoyed working in the VAOL with Brian, Tewa, and Lily and would like to do so again in the future. For the last week of classes we spent a last bit of time analyzing the test videos that we took to ensure the equipment was set up properly and then began the testing that would happen in large amounts for huge sets of raw data.
Brian and I went through the small amount of test videos and quickly recorded anything that the worms did that wasn’t falling normally under gravity.
In the Red and Blue laser set up a worm hovered in the red light at 52 seconds in video 04-04-01, and another worm hovered at 1 minute 16 seconds in the blue light in video 04-04-03. One odd occurrence happened in the videos of dead worms at 3 minutes in the blue light a worm decelerated at an extreme amount which I also believe was caused by a current.
In the Green and Blue laser set up we noticed a worm make a slight turn around at 3 minutes 45 seconds in green light in video 04-07-01, but a current seemed to be the cause as well. However, in video 04-07-02 at 52 seconds a wormed turned around of it’s own accord.Overall, the data seems to suggest that some worms are reacting to the change in light; however, it is not an extremely consistent habit for them to attempt escape from the blue light. Given more time I would like to test the worms under the change into the beam of a UV laser which was mentioned in previous posts. The testing that we began used the same set up and the same methods during the test videos except we set the frame rates on each camera to be the same so that the video speed would remain consistent while analyzing the data in logger pro.
This week, along with taking more data, we also took a cursory look at all the videos so far and simply counted how many worms “turned around” or “hovered” during the data collection. This was just to take a step back and get a sense for what rough results we were getting as to whether the worms have a sensitivity to different wavelengths of light. The results of that:
Red and Blue Lasers
04-04-01 red – 0:52 hovering
04-04-03 blue – 1:16 hovering
04-04-01 blue – 3:00 extreme deceleration (the worm is dead though, so probably due to currents)
Green and Blue Lasers
04-07-01 green – 3:45 (video 1) slight turnaround (maybe due to a current)
04-07-02 green blue video – 0:52 (video 2) turnaround
This data is from 6 (or so) total videos. This data hints that the worms may have a sensitivity to the different wavelengths of light, but is inconclusive so far.
Again, this week we took data with the blue and red lasers.
This image features the beam expander for the red laser, and Brian testing the power of the red laser. It is necessary for the two lasers to be producing the same power output for the experiment to be valid. (Note the setup: in view in order from left to right is the: neutral density expander, the power gauge held by Brian, the beam expander, the tiny cuvette, then the little black square of the CCD camera, and the white screen.)
This semester of research has been so eye-opening and informative to me personally and I feel that it is important to reflect briefly. I have learned through experience that experimental research is not smooth sailing: it requires ingenuity and patience facing the constant barrage of small problems in need of a solution. It is also quite rewarding and easy to invest in– keeping in mind the overall goal of the project, it is fun and easy to fall into the small details and celebrate every small victory. I have found this very rewarding, and am quite excited to continue in the field of research.
In the lab for the past couple of weeks we have been analyzing all of the data we have taken. We did this after we have only taken a few tests mainly to ensure that nothing is going wrong with our data setup so that when we take a massive amount of data it is good to analyze.
Most of the time analyzing the data involves clicking on a video in Logger Pro to track the worms’ movement through the cuvette. As you can see in the picture below, I decided the way I wanted to analyze the data was to have the video from both the red and blue halves of the cuvette on the screen at the same time so that I could track the progress of a worm from one half to the other.
Something odd was happening though where the videos from the blue laser side seemed to take longer to play out and everything seemed to move at a slower pace. Tewa discovered that the frame rate on the blue CCD camera was not set at the same rate as the red; however, a short amount of time in the lab showed that the frame rates can be set on each camera before testing begins, which we will be sure to set at 25 fps from now on
An expression used by experimenters and scientists regarding the collection and arrangement of data; the steps preceding the analysis of the data.
This week, we took data.
The CCD cameras have proven to be excellent. We drop the worm into the cuvette (through which a green and a blue laser are shining). One CCD camera is trained on the green, and one is trained on the blue.
Upload the data to the computer, and open the movies in LoggerPro (Insert-> movie-> choose movie).
“Set Scale” to 1cm by dragging the mouse from one wall of the shadow of the cuvette to the other.
Set origin at the left end of the “set scale” lin
Choose options-> movie options-> override frame rate 25f/s and advance by 15f/s
“add point” and click on the head/tail of the worm. The movie will progress at 15 f/s as you record the entire trajectory
“add point series” for each new worm to separate data and make it easier to look at.
The whole process is not too difficult to understand, and LoggerPro is relatively easy to navigate. The most difficult part is keeping it organized. For each LoggerPro file, there are two movies (one for each of the colors of laser, one from each CCD camera), and two sets of data (two graphs).
Here is a screenshot of the process. There are two videos open, and two sets of data. I keep note of unusual things that happen during the videos, and label the data sets clearly to minimize confusion. LoggerPro also helps differentiate between the data points by changing the color of each point series (each trajectory is color coded).
What’s next? For now, we have to continue collecting data. In order for a set of data to be at all conclusive or valid, it has to have enough raw data to generate a reasonable average.
Although this is not actually week 5 of being in the lab, it is the 5th week of work we have gotten to do. Last week we were unfortunately unable to run any tests due to conflict in timing with the replacement of an air filter in the lab. Although we did not do any testing we did pick worms to put in a new petri dish to grow for the next week.
The first thing that I got to do this week was head into the lab with Tewa and see the finalized version of our test setup. I spent the day taking measurements and drawing a diagram as seen below. This was in the middle of the week and the worms were not mature enough for accurate testing, so the day ended with the measurements and some quick looks at test runs performed by Tewa and Brian the previous week.
The next day was spent in the OLB with Brian and Lily looking for possible UV lasers for testing. After we are done with testing the reactions of the C. Elegans with the blue laser we plan to take it to the next step by replacing it with an ultraviolet laser. It took a lot of looking, but we found a few sites that were selling mounted UV lasers. One sight in particular did not sell mounted lasers, but sold extremely powerful handheld lasers. Although these are dangerous and don’t seem safe for the general populace, the site claims that all of its products are legal under U.S. law.
The final day in the lab we got to run actual tests. The day started out by picking out worms to grow for next week, but the next step was taking them into our actual lab to record some tests. Brian Tewa and I took turns recording each CCD camera and dropping the worms into the cuvette. This new setup is interesting because we no longer have to turn off the lights in the lab to get accurate readings; however, goggles are still needed for safety from the blue laser. The tests seemed to run quite smoothly, and some of the worms seemed as if they were actually fighting against gravity to stay out of the blue light which is what we are looking for. After dropping about 20 worms, the next step was to use chloroform to kill the remaining worms in the petri dish to see if there is a distinct difference between dead and live worms in the cuvette. We walked to a different building to use a fume hood to safely use the chloroform and it took about 10 minutes of waiting to make sure the worms were actually dead. At this point I had to leave the lab, but Tewa and Brian remained to do some more testing.
Next week I will be putting together a table with Brian and Lily regarding the possible UV lasers for Professor Magnes to look at and determine which laser to purchase. I also anticipate more testing with our setup or possibly analyzing the data that we took this week.
Recall the problem of unfocused images when trying to observe the behavior of the worms when sent through two different wavelengths of light, side by side. The regular digital camera was producing such a tiny image that good data was hard to retrieve from the videos. We solved the problem by purchasing a second CCD camera! Now we are able to take two images (focusing the cameras on the two different colors respectively).
What’s next: We are looking into purchasing a UV laser in earnest. Comparing the price, whether it plugs in, the power, the beam divergence, and the wavelength to find the best option for us. We need it to plug in, for the divergence to be small, and for the wavelength to be mid- 300 nm, possibly adjustable.
In our search for the appropriate UV laser, we have found several inappropriate ones, and one especially scary one. A company called Wicked Lasers is selling handheld lasers that achieve up to 2 Watts of power, one of which has an attachment that makes it into a lightsaber. Although it does come with a disclaimer (“this is not a toy”), it is definitely a little scary that such a powerful laser can be bought for just a few hundred dollars, especially one that (even though it is described as NOT a toy) looks an awful lot like a toy.
The CCD camera is not producing images. Up until now, it was possible to set up an effective laser setup just with “eyeballing it” (no math). Unfortunately, the CCD camera needs to be placed a specific distance from the closest lens, and because we have little experience with the camera, some math needs to happen in order for us to develop a “feel” for the instrument.
Due to a whole bunch of work that piled up over the past 2 weeks, I am combining weeks 3 and 4 into one post.
During week 3, Brian showed me a basic intro to analyzing diffraction patterns of c elegans and we spent some time attempting to optimize our optical table. On this blog site there is some data on the diffraction patterns of c elegans and how to analyze them through mathematica. We also watched a video attempting to match the patterns in the video to known patterns on the site to analyze the position and movement of the worms.
On day 2, we went into the actual lab and attempted to analyze data by dropping worms into the split red/blue laser light. Everything was going smoothly until the worms entered the blue light, where we could only see a small blurry circle on the blue image. We repeated our tests a few times and even on the recording you could not clearly see the worms. This was “solved” just before we left the lab by moving the focusing lens for the blue laser closer in the beam path before the final mirror. This change seemed to help immensely with the focus of the image from the blue laser and we could then, for the most part, tell where the worms were.
When week 4 started we once again didn’t have a lot to do because we couldn’t get into the lab and the worms were not ready to be tested. So instead of any hands on testing, Brian showed me how to analyze data from tests he and Tewa ran earlier in the week. Analyzing seemed kind of simple by just using logger pro to view the videos and clicked where the worms are once every 15 frames. You want to always try and click either the head or the tail of the worm each time and not switch back and forth to get the most accurate data set.
Day 2 started with a meeting with Professor Magnes, where she suggested using a CCD camera to record the data instead of the camera now that just recorded the images. We went into the lab after and immediately began setting up the CCD camera to run some tests. We made sure to not place the receptors for the camera in the most intense point of the beam, but the problem was we were not getting the full image that we needed to test. When we moved the camera farther away to get a better view of the image, the receptors stopped transmitting data. We were in a constant battle between trying to find a working distance for the CCD camera to display data, and the distance needed to see the full image we needed to test.
(An image of what the CCD camera was displaying) (Taken by Tewa Kpulun)
Next week we will hopefully get the CCD camera working the way we want it to so that we can record the data accurately. Accurate data is necessary to see if the c elegans have any reaction from the transition from the red into the blue laser light.
In the lab, things do not go smoothly line to line like in classroom physics problems. Experiments take endurance and patience: you will fail before you succeed.
Current project: Analyzing the C. elegans’ reaction to different light wavelengths (blue and red).
Problem: The videocamera cannot effectively record the nematode’s path through both the red and blue– it is very hard to track its path when analyzing the data.
Proposed Solution: CCD Camera.
A “charge-coupled device” camera, although much pricier than a digital camera, converts the light it senses into electrons (just like a solar cell). They then interpret the assembled charge, and transfer the analog data into digital pixels. These cameras produce extremely high-quality images due to the charge moving across the chip with very little distortion.
This particular camera (pictured) is great for the setup because it can 1: be placed directly in the beam to collect images, and 2: plugs into the computer to easily record the images.
What’s next? First we have to confirm that this CCD camera will solve the above problem. Then onto data collection: it is unknown whether the worms react more to green or blue light. It is known that they swim away from UV light. First, collect data for blue and green. Then, on to a UV laser?
This week in the lab we did some new things to prepare for actual data taking which we hope to start as soon as possible. I learned how to measure and record data about the optics table, how to set up/move items on the optics table, how to clean optics, and had some hands-on practice picking worms.
The first day this week, Brian brought in a new string to measure the distances on the optics table so that we could record everything in detail in the lab notebook. To measure the distances between the items on the table, I had to hold the base of the string as close to one item without getting oil from my skin onto it, then pull the string tight and do the same with a point in the middle of the string. Once this distance was measure on the string, it was easy to mark the point on the string with a pen then measure the end to the mark on a ruler. I recorded the data so that Brian could put it into his notebook with the detailed sketch of the table.
When measuring the distances for the blue laser, we came across the problem that the beam from the laser was not traveling exactly where we wanted it to. We spent most of the time after that trying to re-align the setup for the blue laser so that the image off of the mirror is as large as possible. By using the holes drilled into the table you can estimate where the beam will be by trying to have the beam run parallel to the holes directly over top of them.
At the end of that day, Brian gave a quick tutorial on different ways to clean optics. My favorite way is the drop and drag method where you put a single drop of cleaning solution on the cloth and drag it slowly across the optic.
The last major thing I did this week was I got to pick worms for a new petri dish. I thought it would be very difficult to have the worms stay on the pick and not fall off during transfer, but they seemed to wrap around the head of the pick and not let go. It was not too bad picking the worms, and I am sure that I will only get better as the semester goes on.
I am looking forward to next week where we will hopefully finish aligning the blue laser and begin taking data with the worms inside the optics lab.