User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/06/23/Temperature data
Temperature stabilized data
So I was finally able to run an experiment with all the different bovine caseins on my newly temperature stabilized objective. Below are the results.
The orange line is the average temperature for the assay. The grey dots are the average temperatures for each ROI for the assay. There is some rather odd things going on here.
- The whole casein looks good. There is an initial temperature increase due probably to the slide warming up to the objective. I do prepare my slide on my Slide Warmer XJ2 so the most probably cause of this reheating is due to me having to remove the slide from the warmer and move it to the microscope. At any rate, I can get good data that is stable with the whole casein if I don't use the first 10 minutes of data when the slide is on the objective.
- This one is just strange. I have no clue as to what is going on here. I could have been getting close to an air bubble in the slide or close to the tape. As I've found out, I know that the speed at which microtubules glide at is a function of how close they are to the tape of my flow cell. I'm thinking this is the case since the temperature is pretty much stable for this assay.
- More unusual things going on here. I'm thinking that this is just a bad assay.
- There's nothing unusual about this. This is just how microtubules glide when the surface is functionalized with bovine kappa casein.
LED from Bridgelux
This LED can be driven with 900mA of juice. So, I mounted it on the Thorlabs LED holder and put it in the microscope. I was able to get data with it but I did have to change some things.
- Changed the gain to 250.
- This causes dead pixels to become apparent.
- Changed from 5 to 4 frames/s because I needed to change the amount of exposure from 100ms to 200ms.
- Tracking is working with this setup. I'm using the 29% rhodamine labeled microtubules with the LED as well. The only issue is that I can't use auto thresholding. I had to manually set it for 25-255. Everything seems to be working just fine and the results from the experiment will show if I can get away with using the LED or not.
There is one big thing about the LED setup that will be interesting to see. Right now, I made only one sample with whole casein passivation. I used that sample to take data with the LED. I then changed out the Hg bulb (since it was flickering yesterday) and I am now taking data with it. If the speed using the Hg lamp is greater than that of the LED, then I know for sure that the Hg lamp is locally heating my sample and causing the microtubules to glide faster. This should be interesting to see.
So I get fluctuations in my speed measurements in all my assays. I'm curious to see if the cause of this is due to kinesin density on the microscope slip surface. To see if this is the case, I'm looking at a sample in the same spot for 30 minutes (broken up into the usual 2 minutes total exposures) with the Hg lamp.
- I should point out that the sample is the same sample from the LED experiment. I replaced the Hg lamp without removing the sample and started taking data with it after the bulb replacement.
Redo of casein data
It's apparent that I will need to redo the casein experiments. After I'm done looking at the LED and the same spot assays, I'll start the casein experiment again.