Thursday 14 February 2013

Valentine's Day Electron Microscopy

Electron microscopes are pretty impressive machines. By firing electrons at a sample they can generate images thousands, or even millions, times sharper than you can make with light. With the right sample you can spot individual protein molecules in a cell, and even individual atoms within a molecule. When using these microscopes to look at the structures within biological samples like cells there are two big problems though:
  1. Proteins, sugars, fats, DNA, water, plastic, etc. all block electrons by about the same amount. 
  2. Electrons can only travel very short distances through materials.
These two issues make looking at a cell tricky: firstly the cell is too thick for most electrons to travel through it. You can up the power of the electron beam, but then you just vaporise the cell. To solve this the cell has to be sliced up into very thin layers called 'sections'. The next problem is therefore how to slice up a cell, which is basically a bag of proteins dissolved in water. The answer is to replace the water with plastic, making a solidified version of the cell which can be sliced up into thin sections. Finally, to actually tell the difference between protein, fat and DNA in the sample, you need to stain them to make them stand out from the plastic in the background. This is done by using heavy metals, like osmium and lead. The huge positively charged nuclei in these atoms scatter electrons away from the the detector; this makes regions where heavy metals have bound to proteins, fats, etc. look darker.

A block of plasticated cells stained with heavy metals (the black bit) in an epoxy resin plastic (the amber bit) in a metal holder (the silver bit).

So how thin does one of these sections need to be? In short, very. Even travelling through air an electron may only go a couple of centimetres before an atom captures it or deflects it from its path. In a solid material like plastic the distance is far shorter, about 1 million times shorter, with electrons travelling less than 1 millionth of a metre before being scattered or captured. The sections a sample is currently in into must be very thin, about one ten millionth of a metre (100 nanometres) is common.

Cutting a sample into slices one ten millionth of a metre thick is a challenge. It is like cutting a human hair into 1000 slices, each section is about 2000 times thinner than normal office paper. A lab machine called an ultramicrotome is designed to do this, and can cut slices as thin as 30nm. This is a tiny distance, about the width of 15 DNA double helices. The next problem is; how do you know you have cut a slice of the correct thickness? The sections are far too thin to be able to pick them up and measure them by any normal method. Luckily a physical phenomenon called thin film interference gives an easy way to do this.

When light hits a thin film, like a thin layer of oil in water or a soap bubble, some light bounces off the front of the film and some off the back. If the film is a similar thickness to the wavelength of light (around 500 nm) then interference between the two paths the light takes will occur. The interference can be constructive (boosting the light intensity) or destructive (reducing the light intensity). Because different colours of light have different wavelengths, different colours will experience constructive or destructive interference, for example if blue and green wavelengths destructively interfere, but red does not, then only red light will be reflected. The film will look red, even if the material it is made of is totally transparent.

Thin film interference in a soap bubble. CC-BY-SA by link.

Sections of cells embedded in plastic for electron microscopy act just like an oil or soap film, and have distinctive colours based on their thickness. We use these colours to check that the sections we have cut are the correct thickness, without having to directly measure them. It really is very quick and easy!

Lots of electron microscopy sections of different thickness. From bottom to top: 30nm (grey), 50nm (white), 70nm (white), 100nm (gold), 150nm (purple), 200nm (blue), 250nm (yellow), 300nm (pink/red), 400nm (green), 500nm (purple).

So what does this have to do with Valentine's day?

As far as I can work out the sectioning process you use for electron microscopy is a great way to make the smallest possible valentines heart which is still has a vivid pink/red colour. 'All' you need is:
  • An ultramicrotome
  • A block of plastic 
  • A razor blade 
  • Steady hands
  • About 10 minutes
Step 1:
Trim the plastic block to a flat face, then cut a heart shape into that face. This is the bit the steady hands and razor blade are needed for, the heart needs to be about 0.3 to 0.5 mm (250 to 500 um) wide! It also needs to have tidy flat edges so sections can be cut without damaging the knife blade.

The trimmed, heart-shaped, block of plastic.

Step 2:
Use the ultramicrotome to cut a section 300 nm (3 ten millionths of a metre) thick off the block, and catch it on the surface of a pool of water next to the knife edge. From the colour scale of thin film interference we expect a 300 nm section to be a vivid red/pink colour (even though the plastic itself is a transparent amber colour).

A 300nm thick sliver of the block, the tiniest Valentine's heart ever.

Step 3:
Bask in the glory of having made the smallest, and geekiest, valentines heart ever. It is so thin that even if you scaled it up to the size of an A4/letter sheet of paper it would still be 4 times thinner than cling film/saran wrap and 40 times thinner than paper. This tiny thickness gives it a tiny volume: it has a volume of about 3×10-14 metres cubed, and a mass of about 3×10-11 grams. That is similar to the mass of a single, average, human cell.

Software used:
ImageJ: Focus stacking.
Photomatix: HDR image processing.

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