News - April 7, 2011

Cool beauty: the cutting edge of microscopy

Examining the botanical world at micro level in its natural state and not as some shrivelled specimen. This is what the Magellan electron microscope can do, thanks to the unique freezing technique developed for it by Adrian Van Aelst. A whole new world is opened up.

'This is cryo for you!' Microscopist gazes with undisguised pride at the picture on the cover of this issue of Resource. This is what his baby, the Magellan scanning electron microscope, is capable of. This sharpness, this resolution - just try and find that anywhere else: that's the message. Impossible, because his machine is unique. The product looks like an art work and would not look out of place at the current exhibition on the Beauty of Science at the Boijmans van Beuningen Museum in Rotterdam. Van Aelst has been there. 'Although I say it myself, I can do better. I fail to understand why they didn't pop in on us.'
The Magellan is the pride of the Wageningen Electron Microscopic Centre (WEMC). The machine will be officially launched during the Botanical Microscopy Meeting on Monday week. This four-yearly gathering of the world's botanical microscopists is taking place in Wageningen this time. A fantastic opportunity for manager Van Aelst and the Eindhoven-based company FEI, which produces the microscope, to put it centre stage. 

What you see is real
Van Aelst is not exaggerating. The images produced by the Magellan are extraordinary. Yet they are not art, but hard science. To come back to the cover photo for a moment... It is an image of a young leaf of the tomato plant. With a little knowledge of biology, you can immediately make out the pores in the background, and in the foreground you can see the veins on the leaf. The purple-greyish hairs are appendages of the epidermis.  But the indisputable show stealers are the yellow stalks with the four cells on the end of them. 'Gland cells', explains Van Aelst. 'They produce that distinctive smell you get if you rub a finger over a tomato leaf.'
So what is so special about this image? It is not the colours, as they are added later. Electron microscopes (see text box: By feel) do not work with light but with electrons, so the photos are black & white. Van Aelst coloured them in for added effect and contrast. And it is certainly not the enlargement. The section of leaf has only been enlarged by 1,000 times and the Magellan is capable of enlargement by 1.6 million times. No, it is the image itself which is so special: the resolution, the sharpness and the depth. But above all: the accuracy. What you see is the reality. 'This is the actual physiological situation. Nothing has been dehydrated. Nothing has collapsed or been distorted by dehydration: everything is plumped-up and stable. And that is the power of cryo-electron microscopy: you can take a snapshot of reality.'

Living matter
The piece of equipment that can do this is the Magellan, the Cryo-SEM (Scanning Electron Microscope), named after the Portuguese explorer. A better name might have been Amundsen, that of the discoverer of the South Pole. Because the microscope takes pictures of frozen matter - hence the suffix 'cryo', from the Greek word for ice.
It is no exaggeration to say that the Cryo-SEM is Adriaan van Aelst's baby. The 61-year-old researcher more or less invented the machine himself. He does the day-to-day management of the WEMC, housed in the Radix building. The Magellan is the showpiece of the centre's collection. Electron microscopes work in a vacuum, which means that the material you want to examine has to be dry. The processing that is usually required to achieve that (fixation and drying) affects the tissue to be examined. So you are always looking at a distorted version of reality. Cryo electron microscopy bypasses that disadvantage. 'You can look at living material in its natural state. That is a fantastic idea', explains van Aelst. Van Aelst was allowed to purchase one for the first time in 1990. 'One of the first in Europe with that resolution. We have been working with it for years. But little by little you discover that it could be improved on.'
Minus 125 degrees
When the centre moved to radix, Van Aelst seized his chance to ask the boss for a new machine. 'That was the right moment to replace the 18-year-old machine. And besides, I now knew what would be the ideal combination.' In other words: how to get a high resolution at high degrees of enlargement in the cold. 'Theoretically in a warm SEM under ideal conditions a resolution of half a nanometre is possible. With this cryo system I now get the same solution at minus 125 degrees, and that is unique. The machine is so good that you can really show everything in detail down to the last nanometre.'
But this machine still had to be manufactured. The FEI in Eindhoven proved willing to develop it at a reduced price (Wageningen paid considerably less than the standard cost price of around one million euros).
The Magellan creates beautiful botanical images. It can also display the chemical composition of the surface (see text box: By feel). Van Aelst: It is unique in this too. As far as I know, there is not another one like it in the world.' One quarter of the assignments come from outside Wageningen UR. The chip industry, for example, examines new engraving processes with it. Campina asks Van Aelst to examine milk powder and whipping cream. And for Van Akzo he studies paints. Van Aelst: 'In fact, anything you can come up with, we can get you images of it. And you keep encountering new things. That makes microscopic research so exciting: it keeps your imagination busy and never becomes humdrum. And for us it is certainly true that one image says more than a thousand words.'

By feel
A scanning electron microscope draws its images using electrons instead of light. An extremely small (approximately one nanometre) bundle of electrons feels the surface of the material to be examined. Point by point, line by line. The incoming electrons bump into electrons from the atoms on the surface of the sample, which are sucked through a sensor placed above the sample. Depending on the relief on the surface, a varying number of electrons is released. This contrast creates the image. The voltage of the incoming electrons is crucial: the faster the electrons, the deeper they penetrate the specimen. Which is not the idea. The aim is to get a picture of the structure of the surface itself: everything underneath it is distortion. To minimize the penetration of the electrons, low voltages are used (0.5-2 kV). Also, the specimen is coated with an extremely thin film (two nanometres) on its surface. This used to be done with gold or platinum; nowadays tungsten is used. With a tungsten film of this sort, resolutions of up to half a nanometre can be achieved. The corresponding enlargement is then about 1.5 million times. Besides these images of the surface, the microscope also provides additional analytical information. Some of the electrons in the cluster coming in bounce back. These 'backscatter' electrons provide information about the density of the surface material. A third source of information is the x-ray radiation released by the process. The energy in this radiation differs per element and therefore reveals the composition of the material.

Frozen moment
The preparation of a cryo-specimen for high-resolution analysis is a pretty spectacular process. A piece of tissue - leaf, for example - is laid between two tiny aluminium plates of three millimetres in diameter. This sandwich is then placed under enormous pressure (2,300 bar) and frozen in a fraction of a second (two milliseconds) in liquid nitrogen. This is accompanied by a loud bang. It forms amorphous ice so fast that the water in the specimen does not have time to form crystals, which would distort and compress the tissue to be examined. A specimen packed in amorphous ice is literally a moment frozen in time.