User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2011/01/25/Reusable flow cell
This trial will consist of me using:
- The 100W Hg lamp.
- 1.0 mg/mL alpha casein passivation. 10 minute incubation.
- Fluid exchange with 0.5 mg/mL alpha casein with 27μg/mL kinesin and 1 mM ATP. 5 minute incubation using twice as much fluid to passivate.
- Fluid exchange with regular motility solution using 20 μL.
I will take data for about 20 minutes or 10 data points. This allows me to get the slide to the objective temperature with enough stable data points to be able to determine the average speed. Once I have taken this data, the slide will come off of the microscope and a fluid exchange with the same regular motility solution will occur. Again, 20 μL of fluid and 10 data points. I am going to repeat this for a total of 9 times or until I see something catastrophic occur in the data taking.
The 9 times is because there will be 9 different solutions of fluid exchange for the water isotope study. Hopefully my fluid exchanges will not affect the passivation and kinesin adhesion on the slide. But, this is what needs to be determined.
Andy Maloney 12:35, 25 January 2011 (EST): So far there has been no hang ups with the 1st ROI. I will have to move the slide a bit however because it doesn't seem to be totally in focus.
Andy Maloney 13:03, 25 January 2011 (EST): So far everything looks to be working just fine. I did see one issue and that is the objective oil. I'll just have to be careful when changing the fluid so that no oil gets into the chamber. I also decided that I will toss the vinyl strips used to "seal" the flow cell between each fluid exchange.
Andy Maloney 13:10, 25 January 2011 (EST): Hmm, I think I spoke too soon. There seems to be more minus ends lifting off of the surface in the first washing.
Andy Maloney 13:30, 25 January 2011 (EST): No problems with this one. I'm still getting motility.
Andy Maloney 14:32, 25 January 2011 (EST): I was able to get through a third washing but, when I went to try a fourth, the flow cell broke. That's not too bad as the objective oil was starting to encroach on the edges of the flow cell. I need to figure out some method of preventing this from happening.
I think that I'm going to analyze the data I have to see if there are any speed variations with the washings.
Waiting for data
Andy Maloney 16:47, 25 January 2011 (EST): While I wait for data, I've decided to go through another iteration of the resealable flow cell.
One thing I didn't like about the first design is that there is a problem of oil getting into the flow cell. I first tried making a very thin strip of tape that adhered to the edges of the cell. I put the static cling vinyl on it and put it on the scope and really abused the position of the slide so that the possibility of getting oil in it was large. While no oil got into the flow cell, when I removed the vinyl, the tape came with it thus destroying the barrier I made.
As my next iteration, I will try the above but this time I will put a thin piece of vinyl over the tape.
Andy Maloney 17:32, 25 January 2011 (EST): This seems to work even better than before. With the inclusion of the extra piece of vinyl on double stick tape, I have been able to make a barrier to the objective oil to the entrances of the flow cell. Plus, it makes it very easy to clean up any oil that may have come in contact with the flow cell edges. Another great thing about this design is that it limits where the objective can go, i.e. it can't get anywhere close to the edges. This is due to the extra height of the flow cell.
I did do one more iteration of the design. And, that was to make the slip go the entire width of a slide (25mm). This ended up allowing the vinyl to keep in contact with slip and not having to bridge a height change from an 18mm slip to the slide. Now there is just one continuous seam.
I do hope that the data I took today will show that this is possible as the time vested in making one of these slides for an experiment is a lot.