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.

Bright-field leaf sections

I've been trying to get good clean cross-sections of tobacco and Nicotiana benthamiana leaves, and having a few problems with the tobacco cell walls, which have a tendency to rip and tear apart. Following up the suggestions made by a few colleagues I've found that using a different brand of the embedding media, and also sectioning at a lower temperature, makes a significant difference. The section below is 5 microns thick (that's 5 thousandths of a millimetre).

Ironically, I can't get the microtome lab cold enough - these sections were cut at a room temperature of 19 degrees Celsius. I'm planning on moving the microtome to a room with ambient temperature of 12 degrees and trying there.

The cell architecture of N. benthamiana appears to be much more robust and has stayed intact during sectioning, even at a thickness of only 4 microns.

My next stage is to stain the sections.

alien & predator

Certain members of our lab investigate the effects of virus infection on plant 'attractiveness' to insects that act as virus vectors, spreading disease from plant to plant. This means that we can't use insecticides or fungicides to keep down the numbers of other insects that like to nibble and make holey patterns in the leaves. Instead, we use biological control, in the form of tiny predatory mites that feed on the babies of the greedy herbivores.

The mites are tiny and not visible to the naked eye (at least, not to mine). But when I was working on the confocal microscope it was very obvious that the company from which we order our biological control agent wasn't just sending us some sawdust and saying it contained mites. As I was scanning along the epidermal cell layer, I suddenly came across a six legged creature with armour plating down its back, sitting under my lens. I was quite taken aback.

I found a few more on my tissue samples. Normally I rinse the tissue well before imaging, but this time I must not have cleaned the leaves as thoroughly as usual. I quite like the images, they look rather cool. I'm glad it's not a life-size picture though, they remind me of cockroaches.....

more confocal images

I'm using a 'new' GFP construct! It's not newly made, but it's one I haven't used for microscope work before.

Weather forecast on the confocal

The weather is being a bit funny lately. As usual. Sometimes I wonder what we would talk about in the UK if we actually had a more stable climate. Not that we don't have meaningful conversations, but it does mean it's easier to have random conversations with complete strangers on a rather non-controversial topic. Even if the weather behaves for a while, we can still comment on whether the BBC weather forecast has, for some reason got it RIGHT for once (though that's probably just due to the laws of probability - it has to be right at some point. Surely). Or if we'll have a drought this year (it never ever ever has but we still get hose pipe warnings). Or flooding in the summer as soon as it starts to rain again (i.e. two days later).

It was nicely hot and sunny last week, so much so that when the air-conditioning was broken in one of the labs I got visitors whilst I was in the chilly confocal room. Actually, I didn't mind being in the dark and cold for once, although it meant I was missing out on the sunshine outside. For one thing, now that it is officially 'summer' the sun goes down so late that I still got some melanin-inducing rays after work. For another, after sitting in the sun at lunch I was thankful for the cool temperature the microscope has to be kept at.

Now that it's gotten cooler again, I've reverted to wearing a jumper and gloves in there.

'Britain doesn't have climate. It has weather.'

By the way, the whole point of this entry was to show off some of the images I've been getting recently....