Under the electron microscope

[ian]

Nice thing about managing a lab is sometimes you get to have a bit of a play. We had clinical samples to process the other week and also a lab full of slime moulds left over from filming. The chemicals used to get things ready for the EM are extremely dangerous and a pain the bum to handle as we had a sample of Mandy - a wild cribraria, it seemed worth the effort to use the left over chemicals to get some under the microscope.

Slime moulds are damp flexible and quite large, electron microscopes need things that are dry ( we have a hard vacuum and a camera running at -100C - water would cause us much swearing ), rigid and thin enough to get an electron beam through so there's a bit of work to do.

First - fixation - we need our subject to stop being alive and for all cellular processes to freeze into a neat snapshot, for this we use glutaraldehyde which cross links all cell proteins into a solid gel. Glut is fairly nasty stuff.

Living things also have lipids ( oils ), these need fixing and staining so we can see them - chemical of choice for this is osmium tetroxide - in the lab it's one of our deathwish chemicals ( we really do call it that ).

Next we we get the water out using a gradient of acetone, with the water gone we then add epoxy resins and give them a good 24 hours to diffuse in. The samples then get baked overnight at 65C to set the resin.

Then we're finally ready to prepare the sample...

[ian]

Part 2

Two days of work has given us tiny chips of what's basically set glue - it's dry and solid but even though the tissue pieces are just a couple of mm cubed they're too big - we need things nanometers thick for this.

The sample is that tiny black dot in the middle of the yellow resin. It's clamped in the ultramicrotome chuck. The blue object is my diamond knife - mine - nobody else's, we get very precious about our diamond blades - there are only two other people I have ever entrusted it to. The 'tome drives the chuck down drawing the block over the blade, slivers of sample - just 80nm thick float off onto the water where I collect them and load them. You can slice a human hair into a hundred piece with one of these - lengthways.

Staining - cells are basically all carbon and they're set in a resin that's mostly carbon, we need a bit of contrast. The chemicals of choice here are uranium and lead salts. Those tiny discs are the slices of Mandy on copper mesh. After a float and a wash, they're ready to load into the machine

My second favourite toy - a transmission electron microscope

[ian]

Part 3 - the actual pics -

I do a lot of EM work on cells, human samples, cancer cells, plants, bits of cow... I know my way around cellular samples but these things aren't exactly normal and I'm still learning my way around - here's the story so far.

This is not part of my actual work - it's a fun sideline.

A nucleus -surrounded by the thickish double layer of the nuclear membrane.  Home of DNA and control centre of the cell, the darker grey patches inside are nucleoli - areas of active DNA transcription - genes being called into service. Around the nuclear membrane you might just make out the tiny gaps of the nuclear pores where things get in and out

Mitochondria - four of them with their convoluted internal membranes, where oxygen is consumed, sugars are processed into ATP - the cells handy store of energy. Most mitochondria don't look this baroque, crib turns out to have really twiddly cristae ( infoldings ) - never seen anything quite like it. That patch of dark round objects is ( I think ) a bundle of actin filaments - the cells moving stuff fibres responsible for a slimes pulse. In the middle of the patch and in between the two 'c' shaped mitochondria you can also see a couple of internal channels.

[ian]

View through a vein, you can see the smaller channels or possibly just vesicles as small circles around the central part and then the large blobby inclusion of a main vein with - cell contents pickled into fibrous filaments.

And finally - collateral damage - this thing must have been living in the slime layer, I thought it was a bacterium but that dark round object looks like a nucleus ( bacteria don't have them ) and the two oval things appear to be mitochondrial so this is a protist of some description, paramecium might be a likely candidate

[Nemo Andrea]

Wow, very nice Ian! 

Having a look at physarum under the TEM is something I'd love to do, but have not been able to convince anyone its worth the TEM time :).

Do you think any part of physarum would be thin enough as is for inspection under TEM? I don't think I could realistically get it sectioned, but perhaps the end of a retracting protrusion might be thin enough? 

[Kenneth Ramos]

Until now, I always fancied or dreamed of being able to work with an electron microscope.  I never realized however, just how much and what all else there was involved in preparing specimens for observation.  Needless to say, I have learned quite a bit from your presentation that I did not know and I thank you for your time in presenting this post, it was very informative.  In the meantime, I will be content with my Zeiss Axiostar Plus TLM. Great images by the way, very nice.