the last of the fourth year students

Aaargh
I'm officially the last one of my batch of phd students not to have submitted their thesis!! And I haven't even left the lab yet.
I'm hovering between panicking and complacency now that I'm the last one - I can't be any more last than last. Except I know that people who started after me are also starting to write up.

Oh and yes. I finally set up my cantab address. Not that I'll be leaving anytime soon.

Wallace and Gromit rise again

I watched Nick Parks newest Wallace and Gromit short last night - A Matter of Loaf and Death (cue appropriately dramatic and scary music).I love Wallace and Gromit, the writers have a brilliantly wry and British sense of humour - and as my friend points out, they actually create a storyline which works without taking itself too seriously. The amount of attention to detail and the painstaking effort required to film it frame by frame is worthy of our appreciation in itself. How is it that they can convey more expression via modelling clay than most computer-generated animations seem able to do?(excepting some Disney and/or Pixar films - I won't concede all.)
The best bit for me is the waking up of Wallace scene, with a *new and improved* bakery-incorporated method of getting Wallace out of bed and into his trousers (literally). My glee amplified when the contraption played an extra starring role later in the clip.
A bit floury, very nutty and very well done.

What should have happened...

This is what I wanted to achieve with my laser capture microdissection work. This is the cross-section of a tobacco leaf. I've used the laser to remove only the upper epidermal cell layer (The section is upside down). I had to camp out for a week in a room cooled to 12 degrees Celsius, with the cooling motors making a racket in the background, to get these tissue sections.

shooting practice

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.

spoke too soon

It turns out that the end of the light of the tunnel was really just the headlights of an on-coming express train.

I've been trying to put together the figures for my *first* paper (yay). It's a mix of my work and some results from a PhD student who finished last year. My supervisor's writing the main bulk of the paper, which is good because it saves me time, but he's assumed we have images and figures that we don't have, which is BAD. We have the data and some of the photos, but they were meant for our own records and not of publishable quality. The other problem is that the previous student's thesis is a nightmare to trawl through, there are so many different plant lines and he's laid out the results for each experiment in a slightly different format each time (why oh why?!!!) .

Some of these issues I need to bring up with my supervisor, but in true traditional supervisor fashion, he sent me the rough draft on monday and then disappeared for the week on tuesday. I need to give him a mock-up of the figures at the start of next week, and there's been no word from the previous student in response to my cry for help (and data in a more sensible form).

At least I'm lined up to be joint first author on this paper, rather than the second author I thought I might be. But it's a hefty amount of work I need to do to get it.