# Tag Archive: confocal microscopy

May 17 2010

## Understanding mayonnaise

Some time ago I wrote a post on confocal microscopy – a way of probing 3D structure at high spatial resolution. This post is about using confocal microscope to understand mayonnaise (and a bunch of other things)

As young scientists we are introduced to the ideas of solids, liquids and gases very early on. We make these distinctions, amongst other things, to understand their mechanical properties, to answer questions such as: How thick do I have to make the legs of my chair to support my weight? How fast will liquid run out of a bucket? How high will my balloon fly?

But what is mayonnaise? It’s very soft, and can be made to flow but it’s not a proper liquid – you can make a pile of mayonnaise. How do we describe grain in a silo, or an avalanche? In some senses they have properties similar to a liquid: they flow – yet they form heaps which is something a solid does. What about foams –  a pile of shaving foam looks pretty similar to mayonnaise? Starch paste is an even weirder example, it acts like a liquid if you treat it gently but a solid if you try anything quick. (This is known as shear thickening). These mixed systems are known as colloids.

The programme for understanding solids, liquids, gases and these odd systems is to understand the interactions between the “fundamental” particles in the system. For our early courses in solids, liquids, and gases this means understanding what the atoms (or molecules) are doing – how many of them are there in a unit volume, how are they ordered, how do they move and how they interact. Typically there are many “fundamental” particles in whatever you’re looking at so rather than trying to work out in detail what all of them are up to you resort to “statistical mechanics”: finding the right statistical properties of your collection of particles to inform you of their large scale behaviour.

The distinguishing feature of all of our new systems (mayonnaise, grain piles, avalanches, foams, starch paste) is that they are made from lumps of one phase (gas, liquid, solid) in another. Avalanches and grain piles are solid particles in a gas; mayonnaise is an emulsion: liquid droplets (oil) inside another liquid (water); foams are air inside a liquid and starch paste is a solid inside a liquid. These systems are more difficult to analyse than our traditional gases, solids and liquids: firstly their component parts aren’t all simple and aren’t all the same. Particles most likely have different sizes and shapes. Atoms and molecules are all the same size and all the same shape. Secondly, they’re athermal – ambient temperatures don’t jiggle all their bits around to make nice averages.

Confocal microscopy looked like an interesting way to answer some of these important questions about the structures to be found in these complex systems. Mayonnaise turns out not to be a good model system to work with – you can’t see through it. However, you can make an emulsion of different combinations of oil and water, and if you’re cunning you can make an emulsion with over 50% of droplets by volume which is still transparent. Using even more cunning you can make the distribution of droplet sizes relatively small.

Having spent a fair bit of time getting the emulsions transparent with reasonable droplet size distributions, my student, Jasna, came in with some pictures of an emulsion from the confocal microscope: where the oil droplets touched each other the image was brighter, you can see this in the image at the top of this post. This was rather unexpected, and useful. The thing about squishy balls, is that the amount by which they are squished tells you something about how hard they are being squeezed. The size of the little patches tells you how much force each droplet is feeling. So all we have to do to find the force network in an emulsions is measure the size of the bright patches between them.

In the end our work measured the forces between droplets in a compressed emulsion and we found that these measurements agreed with a theory and some computer simulations. Criticisms of the work were that the relationship between luminous patch size and force was more complicated than we had assumed, and that the force distribution was all very well but the interesting thing was the arrangement of those forces. These criticisms are fair enough. Must have been pretty good though, because someone wrote a paper for Science claiming to have done it first, whilst citing our paper (they had to publish a correction)!

Footnotes
This work can be found in this paper:

Brujic, J., S. F. Edwards, D. V. Grinev, I. Hopkinson, D. Brujic, and H. A. Makse. “3D bulk measurements of the force distribution in a compressed emulsion system.” Faraday  Discussions 123, (2003), 207-220.  (pdf file on Scribd)
Jasna Brujic was the PhD student who did the experimental work, Sir Sam Edwards is a theoretician who works on granular materials, Dmitri Grinev worked with Sir Sam on the theory, I supervised Jasna, Djordje Brujic is Jasna’s dad and wrote the image analysis code and Hernan Makse is a computer simulator of granular materials.

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.