User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/08/05/Casein experiments
I finished all my work in regards to the temperature stabilization of the objective. It now reads out temperature quite well and tags the files with time stamps in the same manner that Live Feed does. I also got the other PI controller from TeTech up and running, although not as well as I want it to. It is right now working as my slide warmer.
Previously, I didn't exactly know what the temperature of the objective was due to a very poor setup. This caused me to put the temperature of the objective higher than was necessary for it to be at 30C. It was something like 32 or 33C. The slide warmer is at 30C and has always been so. In my data, I saw a characteristic "warm up" time where the data I took was not very stable until 10 minutes into data taking. I'm hoping to see a much less "warm up" time since my slide is at 30C and my objective is at 30C.
So this is the first experiment to determine if everything is working. This casein was not frozen and I'm using 0.5mg/mL whole casein with the kinesin and passivating the surface with 1.0mg/mL. I plan on doing this with the other casein constituents so I figured I may as well try it with the whole casein.
So there are some things to note about the first assay.
- I kept getting MTs disintegrating early on. Now they are moving fine and everything looks great. It may be due to the fact that I had the Hg lamp at 50% but I'm not sure it is.
- I cannot get the entire FOV to be in focus. This may be caused by the top thermometer. Not sure and I will have to look at it once I'm done taking data.
- The FOV is going out of focus. This is a big thing since I know for a fact that the reason it goes out of focus is because of temperature fluctuations. I now have my objective set temperature at 30.3C which may be too low for it to combat the affects of the Hg lamp. I'll have to look at the data and see but for now, I'm going to have to constantly monitor the microscope.
- Hmm. I just saw a squiggle. This may because of the increased casein in solution. I wonder if passivating the slide first is necessary. I wonder if the kinesin bind to the casein before being introduced to the slide.
- Movie 08.
- I'm also seeing really tiny buggers exhibiting motility. And when I mean tiny, I'm talking 1um.
- Movie 09
- There is one with a tail.
- So my temperature is less in this experiment. This means that the gliding speed will be less as well. But, What if I did it at the 32.3C temp? Presumably, if there is more casein on the slide, this means that there may be more kinesin stuck to the casein. That would slow down the speed as well. I think I'll try that.
I was hoping that the MTs would last long enough to try the thing mentioned above. However, I think my Taxol is bad so the MTs just blew up.
Andy Maloney 23:32, 5 August 2010 (EDT): Update
- As expected, the speed is lower at this temperature than at 32C.
- Also, it looks like the Hg lamp is heating up the sample at 30C so I will need to keep it at 32 or higher. It's not that easily seen in the pic but, it definitely is not flat like the other data.
- Maybe I should detach the Hg lamp from the microscope.
- I still don't know if the speed decrease is due to the increase of casein on the surface or the temp but I'm pretty sure it's the temp. It's a big difference though between 2˚C. Something like 200 nm/s.
The reason why I couldn't get the entire FOV to be in focus is because of the way I mounted the top thermometer. I used tape to get it to stick and since the objective is an oil immersion objective, oil seeped underneath the tape. This cause the tape to curl up and move the slide. I normally hate beyond all comprehension to glue things but I cannot think of any way around this one. So, I am now using some thermal epoxy to glue the thermometer down to the top of the objective. I reasoned that since we already have an identical objective to the one I am using right now, and that one is not in use, I could play around with the one I have. So, if I totally destroy it. 1st off, sorry. Second, we have another one so I don't have to be delayed with experiments.