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Dec 03 2009

Confocal microscopy

Back to stuff I should know, in theory, at least.

I had my first microscope as a child, it was a small one but I had a great time looking at little creatures that lived in dirty pond-water. I remember spending a long afternoon trying to see transparent single celled animals, and finally getting the lighting just right to see an amoeba. I also remember trying to immobilise a tiny worm with white spirit – it exploded, but I like to think it died happy.

This week I shall mostly be talking about ‘confocal microscopy’, this is a type of light microscopy (as opposed to electron, infra-red, x-ray, scanning tunnelling, or atomic force microscopy). The smallest thing you can see with a light microscopy is about 1 micron across, that’s a thousandth of a millimetre – a human hair is about 80 micron in diameter. A normal light microscope gives you a nice focused picture of a slice of you sample at the “focal plane”, but it also lets in loads of light from parts of your sample away from the focal plane which leaves you, overall, with a bit of a blurry picture. Microscopists get around this problem by slicing their samples up very thinly hence no bits to be blurry, but this is a fiddly procedure and leaves your sample very dead even if it started alive.

Confocal microscopy is a technique by which the slicing of the sample happens virtually, you can put a big fat sample in the microscope and by the use of cunning optics you only get an image from the focal plane which is lovely and sharp. You can build up a 3D picture of the sample by moving it up and down in front of the lens. Marvin Minsky was the original inventor of the confocal microscope in about 1955 but was somewhat held back by the lack of lasers, computers and stuff. Things picked up again in the 1980’s as these things became readily available. Oops, I think that might have been some cod history ;-)

An interesting feature of the confocal microscope is that if there’s nothing in the focal plane, you don’t see anything (unlike a conventional light microscope where you can always see a big bright something, even if it’s blurry) this can be disconcerting for the learner – you can’t find your sample!

Every microscopy needs a contrast mechanism, a way of separating one thing from another. In confocal microscopy by far the most popular contrast mechanism is to use fluorescence via the use of a fluorescent dye to label bits of your sample.  If you illuminate a fluorescent dye with light of one colour it emits light of another colour (making it stand out particularly well). If you ask an organism nicely (okay – genetically engineer), you can get it to make Green Fluorescent Protein (GFP) which is a protein that fluoresces green (duh!).  All that remains is to find a way of  sticking the fluorescent dye to the thing in which you’re interested.

In each post about science I like to add a little fact to help you wind up / avoid winding up practitioners in that field. So to wind up a microscopist: project an image onto a screen for a presentation and claim “x800” magnification (or whatever). The problem is: to what does “x800” magnification apply? Is it what the microscope told you when you looked through the eyepiece? Is it the magnification on the printed page, the computer screen or on the wall? We really doubt you know. It’s scale bars all the way.

For several years I was proud keeper of a confocal microscope. I, and my students, had great fun with the microscope and it had fun with us. The pointy end of the microscope is the objective lens, the bit closest to the sample. A fancy microscope like our Zeiss LSM 510 had 5 or more objectives mounted on a turret (see the image at the top of the post), each objective gives different magnification. The Zeiss LSM 510 was fully motorised, and too clever by half. It would assume that you wanted to stay focused on the same part of the sample when you changed objectives (or it changed them for you, with it’s motors). Now the problem is that for a x10 objective the focal plane is about 1cm from the front of the objective lens, and for a x40 objective lens it could be only a tenth of a millimetre. Now imagine I’ve just focused deeply inside my sample using an x10 objective, I switch to the x40 object on the computer….. and the microscope mashes the x40 objective lens into the sample, blithely ignoring the sound of £6000 lens smashing glass coverslip and covering it in sticky sample!

In later posts I’ll show some of the results from the confocal microscope in non-mashy-lens-into-sample mode.

Here are some images, these are all slices through solid objects. I didn’t really think this through in terms of explaining what’s in these first three images, roughly they’re what you get if you add a small amount of salt-water to Fairy liquid (although I would prefer you to use Persil washing up liquid). First up is a cross-section through an “onion-type micelle”:

And these are the structures you see in a similar system but with a different concentration of water:

This is a false colour image, bit lurid – don’t know what I was thinking at the time. These are known as “myelin”:

Pollen-grains are always popular – I stole this one from here. Each of the images is a slice, and the inset bottom right is the result of adding all the slices together.