Swammerdam Institute for Life Sciences
Focus on research: microscopist Erik Manders
Erik Manders, microscopist at the Centre for Advanced Microscopy (CAM) in the Swammerdam Institute for Life Sciences (SILS), has devised a way to keep cells alive under an optical microscope while he examines them. A smart on/off light switch for the confocal microscope restricts the amount of light it emits, light that is lethal to cells. His invention, Controlled Light Exposure Microscopy (CLEM), which has meanwhile been marketed by Nikon, has led to an important publication and, according to Manders, CLEM could become very popular in the microscopy field within the next ten years.
When Erik Manders was a postdoc at Oxford in the nineties, it wasn’t yet wholly acknowledged that a microscope’s glaring laser light was such a determining factor for making an image of a living cell. He wanted to examine how chromosomes, which contain hereditary material, move in the nucleus during cell division. ‘It was well-known that each chromosome occupies its own territory within a cell’, says Manders. ‘However, nobody knew how exactly a chromosome was folded up within its territory. I knew that I would be able to discover this if I was able to film a chromosome during cell division.’
Light damage
‘When filming, I used a confocal microscope with which three-dimensional images can be made. The best way to do this was still unknown then: practically nobody did live cell imaging in those days. I turned on the microscope and waited, but nothing happened. The cell I was observing didn’t go through mitosis (cell division), while all other cells in the specimen did. And it kept on going wrong. At first I thought that I was just unlucky, later I discovered that I stopped cell division myself: the light I used to observe the cell had a toxic effect. I reduced the amount of light, and kept on reducing it further, convinced that it must work somehow. I was really fed up when I went home each evening. But one night it worked, I had a cell in which I was able to follow the process of cell division. I got my film, and was at last able to describe how a chromosome is folded up. The conclusion was that the DNA is stored in the nucleus, all neat and tidy – a necessity with 2 metres of DNA in a nucleus measuring 10 micrometers! Compare it to 10 km of kite string in a tennis ball: if that isn’t tidily rolled up, it would be a mess!’
Confocal microscope
In a normal microscope, part of an object is clearly focussed, while that part of the slide not in focus is seen as a fuzzy background. By means of an optical trick, a confocal microscope only shows what is clearly focussed. In a confocal microscope, a laser beam scans through the fluorescent specimen. A three-dimensional image of the specimen is made by moving the focus through the specimen, registering precisely the amount of light received. By repeating this 3D scan every few minutes, a three-dimensional film is formed (4D-imaging of living cells). The confocal microscope was thought up by Marvin Minski in 1961 but because there were hardly any lasers or computers at the time, it wasn’t possible to fabricate it. The first confocal microscope was eventually built in 1979 by Professor Brakenhoff (who also works at CAM).
Signal noise
Later on, Manders went to work in Dorus Gadella’s group where advanced microscope techniques are used to study living cells. He kept looking for ways to reduce the amount of light as much as possible. The photons (light particles) in the laser turn on the fluorescent substances in the cell and this fluorescence creates oxygen radicals which damage and ultimately destroy the cell. ‘The fewer the photons, the less light damage, but the result is a very ‘noisy’ image. You needed to weigh up the pros and cons: either you get an image with a lot of noise, but of a living cell, or you get a good image of a dead cell,’ says Manders.
Manders wanted both a good image and a living cell. He eventually thought up a new way to create images. ‘We are used to a way of creating images whereby each object is illuminated by the same amount of light. Look around you: every object you look at is illuminated by approximately the same amount of light. Yet you don’t need uniform lighting to create images. I turned the way of imaging upside down. Suppose you have a weakly fluorescent object beside a very strongly fluorescent object. Very few photons are obtained from the weak object, so that stronger illumination is required in order to get sufficient photons for a good signal-noise ratio. But the bright object doesn’t need that amount of light to give a good signal-noise ratio and the background, between the objects, doesn’t require any illumination at all, as there’s nothing interesting to be seen there.
‘So there are three different sorts of pixels: the uninteresting background pixels, the weak foreground pixels that you would like to see clearly, and the really bright foreground pixels, that you can also see with less light. And so I thought, if I illuminate each pixel separately, I can regulate the signal-noise ratio in my own way. In fact, I illuminate the specimen very selectively and therefore require only a small amount of light in order to see the same thing: to take a surreptitious look at the cell. Super-simple, but also super-effective!’
‘Compare it to somebody breaking into the bank and looking for the safe. A stupid burglar turns on all the lights in the whole bank building in one fell swoop. This burglar eventually finds the information he’s looking for, the safe, but will pay for the damaging influence of all that light. A clever burglar walks about the building with a torch and uses his light selectively. He finds the safe too, but with much fewer detrimental effects from light! That’s how CLEM works.’
Manders built the first CLEM model together with Ron Hoeve and Carel van Oven, both of whom work at the AMC: an electronics box that measures the light in the pixel and turns it off as soon as it has obtained sufficient photons. CLEM calculates the amount of fluorescence on the basis of exposure time and the number of photons measured by the box.
Patent
CLEM works. Thanks to the reduced exposure time, cells remain alive seven times as long and the image quality isn’t affected negatively. Nature Biotechnology published the article on CLEM, and Manders submitted a patent request, first only in the Netherlands, then in Japan, America and Europe. The Japanese company Nikon now produces CLEM and will market it in Europe in April. ‘I see an advertisement for CLEM now and then in scientific journals on microscopy. That is, of course, very satisfying’, says Manders enthusiastically. ‘Moreover, as a scientist it’s nice to be involved in a commercialization process like this, something that doesn’t happen very often at the UvA compared to the technical universities in the Netherlands or compared to American universities.
Manders has obtained a grant from NWO for a new microscope. ‘It’s rather amusing that we will be buying our own invention from Nikon. We want to have a super set-up for studying living cells, with various lasers and all the newest gadgets. It’s amazing to discover what’s possible nowadays. A microscope shows you things that you don’t normally see. As a child, I used to look at water beetles under the microscope. It takes you into another world and, the more technology, the more you can see of that world, ever more details and information.’
‘In my opinion, CLEM could turn out to be very important. It’s quite possible that, in ten years time, all confocal microscopes are equipped with CLEM’, predicts Manders. Besides publication, patenting and commercialization of scientific knowledge, Manders is of the opinion that media exposure is also very important. ‘The grant for the microscope is financed by tax-payers. They have the right to know exactly what we are going to do with all that money.’