Prep Work Continued
- Making flow cells continued:
- Dried off cover slips. They are very cloudy, but at least that means something's sticking I guess?
- Putting together flow cell notes:
- Tearing off tape - pick up tape by the side since that will get cut off later anyway, tear off at the mark left by the roll
- Putting on tape - put on two pieces leaving about 5-6 mm between the two sides
- Smashing tape - Can use the nitrogen to blow down the tape or use side of the razor blade (don't slide the razor blade, that doesn't work very well, try to press evenly so slide doesn't crack at one place)
- Next cut the edges off from the tape - can use device but really just easier to use the razor blade, make sure the edge of the tape is cut really well because otherwise it won't peel off
- Now use tweezers to position cover slip on its side at the edge of where the flow channel will begin, then let it fall or if it sticks, kind of encourage it with the edge of the tweezers
- More smashing of the tape, this time over the cover slide - need to press hard, but slip is fragile, so be careful at the edges where it isn't over the tape or just in general don't press too hard just in one place
- Flow cell is finished, put back in the container and will clean off with PEM before using, so hopefully the cloudiness will not interfere with anything
Estimate on Number of Liposomes Needed for Flow Cell
- How big is the area we see in the microscope?
Entire slide is 25 by 75 mm
Cover slip is about 20 by 20 mm
Flow cell area approx 18 x 5 mm
About 100 nm diameter
- How many liposomes are needed to completely cover the area?
Let's say for a single layer in flow cell, so divide up 18 x 5 mm by 100 nm
9 * 10^(-5) m2 / 100E-9 m = 900
So would need ~900 liposomes?
- What is concentration in number of liposomes per microliter?
The normal concentration is 2mM or 2E-3Mol/liter
- As an example the number of lipids in a 100 nm size liposome of pure PC is about 80,047 (Ntot)
- This gives the number of liposomes per milliliter
- For a 2mM solution, 100 nm size liposome that is mostly PC
- That would be 1.5 E10 liposomes per milliliter. Per microliter would be dividing by another E3, or 1.5 E7 liposome per microliter
- That is a comparatively HUGELY big amount when you consider we flow through about what, 10 microliters?
- What concentration should be flowed through the cell?
OK, so assume we are going to put through . . . 10 microliters?
Solving for concentration I get:
Mlipid = (Nlip x Ntot x 1000/NA) (/ 1000) (* 10)
Mlipid = (80047 x 900 x 1000/6.022E23) (/ 1000) (* 10)
Mlipid = 1.2 E-15 M (That is tiny! I don't think I can accurately measure volumes well enough to get to this molarity)
- Just for interest's sake, let's say I was starting with 2 mM and want to get to 1.2 E-12 mM
2 E-3 mol / liter *V1 = 1.2E-15 mol/liter * 1E-3 liter
V1= 6 E-16 liters or 6E-10 microliters which is really small