The optical tweezers apparatus is finally functioning, so I spent yesterday trying it out. I can't yet do what I want to do, but enough of the steps are working that I've been able to get to a new problem.
The various optical components have been adjusted, so now I could (sometimes) trap beads at the laser focus. This is quite fussy; most of the time the beads are pulled into the focus point (the trap) and then spit right out again. My colleague thinks this is probably because of a bit of astigmatism in the (cheap) laser, and because she's now using an oil-immersion lens rather than a water immersion lens. I don't understand why the oil immersion lens would be worse, as the oil is specially designed to have the same refractive index as the glass on either side of it (the objective lens and the cover slip) so oil causes less refraction than water. But anyway, the trap works best when it is within 20 microns of the coverslip/oil/lens. Unfortunately it's tricky to get this distance because the micrometer used to move the chamber forwards and back has quite a bit of wobble ('hysteresis').
I could also focus on the cells I had attached to the coverslip, and the images looked much clearer than they had before the optics were adjusted. Again though, the wobble in the micrometer made this somewhat imprecise. And once I'd trapped a bead I could use the micrometer to move the chamber away from the objective, thus bringing the trap position to the surface of the coverslip where the cells were.
At this point, if the beads had DNA on them, the cells might attach to the DNA. But the beads I had brought were too small. I had made them in a bit of a rush the night before, and although I was quite sure I'd taken the beads from the little bottle labeled "2.1 micron" I must have somehow used the one labeled "1.2 micron". So I was using 2 micron beads without DNA, taken from an old tube that was in the drawer of the tweezers lab.
But this is where the new problem became evident. When I brought the beads to the coverslip where the cells were, the beads quickly became stuck to the coverslip. This wasn't exactly a surprise. I knew this was likely to be a problem, and had been trying out ways to prevent it a couple of months ago. But I hadn't found any that worked. So I need to go back and try some more, both treating the coverslip after the cells have attached, and pre-treating the beads after the DNA has attached.
I may also be able to solve this problem by not petting the beads touch the cover slip in the first place. In principle I should be able to bring the bead to a position that's still a few microns above the coverslip. Depending on how far the DNA extends from the bead surface, the cell will be able to contact the DNA but the bead will not contact the coverslip. The problem here is the poor control of the chamber position (the wobbly micrometer). My colleague has what appears to be a better micrometer (bought on eBay!), but she's not sure it is compatible with the present setup.
I'm going to make one more try at this before submitting the CIHR grant (due in a month). This time I'll take more time to carefully prepare my 2.1 micron beads with attached DNA. The problem of beads sticking to the sides of the tube when being washed hasn't been solved but it can be minimized. I'll add biotin to the first wash to block the streptavidin, and finally resuspend them in TE with added BSA to reduce nonspecific binding.
Welcome to the 4th Reich part 1.
4 hours ago in Angry by Choice