Robert Hooke was a pioneer of microscopy, when back in the 17th century he drew stunning images of insects, plant cells and fossils. Since then microscopes that use light to magnify things we can’t see with the naked eye have, of course, improved. But, surprisingly, 300 years of engineering lenses hasn’t improved things all that much.
There happens to be a fundamental limit on the magnification that can be obtained from light microscopes. This limit was defined by Ernest Abbe in 1873. The Abbe limit says that we won’t see things smaller than half the wavelength of light.
The wavelength of green light is about 550nm – where nm stands for a billionth of a metre – so practically speaking, anything smaller that about 250nm is below Abbe’s limit. Hooke and other microscopists are fine with fleas (1mm is about 5,000 times the limit), hairs (100 micrometre, about 500 times the limit) and animal cells (50 micrometres). But no matter how good you make a lens you would never be able to use light to get sharp images of bacteria (500nm) or see images of viruses (100nm), proteins (10nm) or small molecules (1nm).
That is when microscopists turned to electrons. The resolution achieved by using electron microscopes was about 100pm – where pm stands for a trillionth of a metre. The result is that electron microscopes can see objects 2,000 times smaller than Abbe’s limit.
But there is a trade off. Electron microscopy requires the sample to be under a high vacuum. The result is that the material you want to visualise can’t be alive. Observing any real-time biological process, then, is out of question.
The 2014 chemistry Nobel laureates, Eric Betzig, Stefan Hell and William Moerner, saw Abbe’s predictions as less of a limit and more of a challenge. Their pioneering work used fluorescence to circumvent the limit and turn microscopes into nanoscopes.
Fluorescence is the process where a chemical absorbs light of one colour and then emits it as another colour. It crops up in washing powder, for example, where a compound absorbs ultraviolet light (that you can’t see) and emits a dim blue light (that you can see). In the process tricking you into thinking your wash is cleaner than it really is.
Scientists use fluorescence to tag molecules of interest. So they might add a fluorescent marker that can stick to DNA. Then, under a microscope, they observe shining molecules which tells them approximately where the DNA is. But this doesn’t get around Abbe’s limit. They only see a blob of brightly coloured material in the cell, which is to say that resolution of the image remains limited by the wavelength of light used to visualise it.
Standing on others’ shoulders
This year’s Nobel Prize in chemistry perfectly shows how scientists build on each others’ work to do greater things than the sum of each person’s individual contributions. It also celebrates the importance of interdisciplinary work, because the three laureates include a physicist, a chemist and a biologist.
Hell’s innovation was to come up with a way of suppressing the light from most of the blob. This leaves a tiny, glowing, nanometre-sized area of interest. He used one laser to excite and fluoresce the molecules, and a second to turn them off again, except in the area of interest.
The result is that light is only emitted from a volume far smaller than Abbe’s limit and the resolution of the image is improved many times. The process is then repeated while scanning over a sample, giving a clear picture of a virus or bacteria. This technique is now known as stimulated emission depletion (STED) microscopy.
Moerner and Betzig took another approach, by building on the Nobel Prize-winning discovery of Green Fluorescent protein (GFP) in jellyfish. When GFP is illuminated with blue light it glows green, just as fluorescence predicts. This turns out to be very useful. Much like tagging DNA, GFP could be fused with another protein and seen under a microscope. However, if you keep shining the blue light on GFP it fades, limiting the time you can visualise the sample.
Moerner discovered that GFP has a perfect recovery switch. Once the glow has faded away it can be reactivated by illuminating with near-UV light (at 405nm), allowing it to fluoresce anew. Moerner went on to disperse GFP in a gel and then switch individual molecules on and off, becoming the first person to detect a single molecule with light microscopy. And this discovery was just what Betzig was waiting for.
Betzig targeted GFP to particular areas of a cell. He then illuminated the GFPs with very weak blue light. Consequently, just a few molecules were excited enough to glow, but they could each be clearly seen, he took their picture and allowed their light to fade away. Then he repeated the process, but this time a different subset of molecules glowed, were photographed and faded. He repeated this many, many times and then superimposed the images. The resulting image had a resolution far below Abbe’s limit.
Calling Betzig, Hell and Moerner’s innovations high-resolution microscopy is a misnomer. Their work has allowed scientists to study processes far below the microscale (thousandth of a metre) to the nanoscale (billionth of a metre). They have produced “nanoscopes” that allow us to investigate the processes going on in living organisms in real-time.
Hell has used his technique to investigate how nerve cells transmit messages around the body, Moerner has studied the proteins involved in Huntington’s disease and Beitzig has probed cells dividing in an embryo. But, more importantly, these scientists have given us tools that many can use to probe other biological processes, which couldn’t be studied otherwise.
The 17th-century scientist Antonie van Leeuwenhoek is called the father of microbiology, because it is said that for the many decades that he worked he saw things under his microscopes that no human being had ever seen before. The winners of the 2014 Nobel Prize in chemistry have done something similar. Their microscopes are revealing images of things that always existed but which we were never able to see before in such detail.