I spent a very frustrating couple of hours on the laser capture microdissection machine on Saturday. The aim was to capture a sample of about 250 cells from a fixed leaf section of 5 microns thick and extract the RNA from them, to use for RNA analysis. The basic idea is very simple - you have a 'cap' of thin plastic film which you direct the machine to position above your tissue section. You can then look at a microscope image which is projected onto your computer screen and mark which individual cells you want to capture. The computer then relays this information to the laser arm which fires a laser beam for a period in the microsecond region at the cap above each point. The film then melts, adhering the cell as it reforms, so that when you lift the cap off the desired cells should be adhered to the film.Very cool, and I've had brilliant results from it. The hardest part is only really optimising the laser parameters, as it has to be strong enough to melt the film, but not so strong that it burns a hole in your tissue. However, it seemed that the laser just didn't want to play on Saturday. That or it was too eager. It jostled between not melting the film, only making dents in the surface (see top image) or burning a black hole in the middle of the target point. As the RNA in the tissue section only remains intact for an hour or so after wax deparaffinization, by the time I had the laser optimised there was no time to capture the cells I wanted.
Not yet defeated by this failure, I prepared a second section, and set about re-optimising the laser for this sample (you have to re-set everything each time you change samples). Again it wasn't playing. My cap looked like it'd been used for target practice.
In the end I gave up. It was only when I was removing my caps from the machine and complaining to a friend that he noticed a dust-hair on the surface of the cap. Those very fine ones that float about. But it was enough to prevent the cap from sitting flush against the tissue surface - hence my problems with the laser. Sigh. At least now I know, so I'll be cleaning the cap holder very well before using it in the future.
2 則留言:
ok ok. the part where you said 'the idea was very simple'.... i didn't get the rest of it.
aaarrghh
basically you use a laser beam to melt some plastic onto your cells. Then you let it reset, lift off the plastic, and your cells should be stuck to it.
If the laser isn't hot enough, the plastic doesn't melt, so no sticking.
If the laser is too hot it burns a hole in your cells.
better?
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