By Beth Mole and Meghan Rosen
For more than a century, researchers have observed very tiny objects using the light — or optical — microscope. But there’s a limit on how small an object such scopes can bring into focus. In 1873, Ernst Abbe calculated that limit, called a diffraction barrier. It was 0.2 micrometer — or half the wavelength of visible light. And that limit held until very recently, when three scientists developed ways to see things far smaller than that. For breaking through that diffraction barrier, they will now share the 2014 Nobel Prize in chemistry.
Their advance is known as fluorescence microscopy. The key to seeing features on the nano — or billionth-of-a-meter — scale is using molecules that fluoresce, or glow.
On October 8, physicists Eric Betzig of the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Va., Stefan Hell of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, and physical chemist William E. Moerner of Stanford University in Palo Alto, Calif., learned they will split the roughly $1.1 million chemistry prize.
“The scientific community wasn’t very receptive to the idea of overcoming the diffraction barrier,” Hell said at a press conference. The barrier had been around for more than a century. People believed “that doing something about it was — pardon me — kind of crazy,” he recalled.
But the new technique did, in fact, succeed.
“It has provided a window into the cell,” says biophysicist Catherine Lewis. She directs cell biology and biophysics at the National Institute of General Medical Sciences in Bethesda, Md.
The technique lets people peer into the depths of bacterial cells or watch nerve cells shift shapes in learning brains. It even allows people to glimpse the clumped proteins that underlie disorders such as Alzheimer’s, Huntington’s and Parkinson’s diseases.
The science behind it all
If traditional microscopy is like looking at an anthill, fluorescence microscopy is like looking at individual ants — only better, says Sven Lidin of Lund University in Sweden. This chair of the Nobel Committee for Chemistry spoke at a news conference.
A human hair is about 500 times larger than the limit of fluorescence microscopy, he noted, as he plucked a strand from his own head. So conventional microscopes can easily see hair-size objects. But most of the machinery and features of living cells are a hundredth or thousandth of a hair’s width. They are far smaller than what a regular microscope can bring into focus.
When Hell and his colleagues looked at cell parts with a light microscope, those features all bunched together. They just looked like a blob. But, if scientists could focus on just one part at a time, they might be able to see details inside the blob, he recalls thinking. In the end, he, Betzig and Moerner succeeded “by playing with the molecules,” Hell says.
In 2000, Hell’s team beamed light from a combo of colored lasers at fluorescent molecules. The first laser lit up a wide group of molecules. At the same time, a second laser — with a donut-shaped beam — knocked out the outside ring of light. This left a tiny circle of molecules spotlighted in the center. They could be seen and they were smaller than 0.2 micrometer in size. This meant the researchers had surpassed the lower limit on how small a feature the light microscope could see.
Other researchers have devised a different way to spy on such wee molecules. It’s called electron microscopy. To use it, Lidin notes, scientists must first kill cells. Then they have to slice them, stain them with chemicals and blast them with intense radiation. With fluorescence microscopy, researchers can observe bacteria without destroying them, Lidin says. “They can be studied in real time while they live and prosper.”
To watch the daily happenings inside cells, Moerner and Betzig used the same microscopy trick as Hell, but with an added twist. They didn’t block light from neighboring molecules. Instead, they engineered molecules to have light switches, explains Brian English. He, too, works at HHMI’s Janelia Research Campus. Three years before Hell’s team, Moerner’s group showed that zapping fluorescent molecules with different wavelengths of light could cause individual molecules to light up or black out.
Building on that finding, in 2006 Betzig and his colleagues used similar molecules to view a lone membrane protein from a mammalian cell. The method, he says, could one day uncover how molecules “come together to create animate life.”
Chemist Richard Zare of Stanford marvels at the advances. “We all in chemistry — we believed in molecules,” he says. “But no one had ever seen one” before these techniques, he says.
Alzheimer’s disease An incurable brain disease that can cause confusion, mood changes and problems with memory, language, behavior and problem solving. No cause or cure is known.
bacterium (plural bacteria) A single-celled organism. These dwell nearly everywhere on Earth, from the bottom of the sea to inside animals.
cell The smallest structural and functional unit of an organism. Typically too small to see with the naked eye,it consists of watery fluid surrounded by a membrane or wall. Animals are made of anywhere from thousands to trillions of cells, depending on their size.
chemistry The field of science that deals with the composition, structure and properties of substances and how they interact with one another. Chemists use this knowledge to study unfamiliar substances, to reproduce large quantities of useful substances or to design and create new and useful substances.
diffraction The bending of waves when they hit an object. The pattern produced by those waves can be used to determine the structure of very tiny objects, such as the width of a human hair.
E. coli (short for Escherichia coli) A bacterium that researchers often use to study genetics. Some types of this microbe cause disease, but many other forms of it do not.
electron microscope A microscope with high resolution and magnification that uses electrons rather than light to image an object.
embryo The early stages of a developing vertebrate, or animal with a backbone, consisting only one or a or a few cells. As an adjective, the term would be embryonic.
engineering The field of research that uses math and science to solve practical problems.
fluorescent Capable of absorbing and reemitting light. That reemitted light is known as a fluorescence.
Huntington’s disease An inherited disease, Huntington’s is caused by a mutation in a gene called huntingtin. People with the disease develop memory problems, mental illness, uncontrolled writhing movements and other movement symptoms. Subtle symptoms usually first show up in middle-adulthood. Over time symptoms become more severe. Huntington’s disease affects between five and 10 out of every 100,000 people.
laser A device that generates an intense beam of coherent light of a single color. Lasers are used in drilling and cutting, alignment and guidance, in data storage and in surgery.
membrane A barrier which blocks the passage (or flow through of) some materials depending on their size or other features. Membranes are an integral part of filtration systems. Many serve that function on cells or organs of a body.
microscope An instrument used to view objects, like bacteria, or the single cells of plants or animals, that are too small to be visible to the unaided eye.
molecule An electrically neutral group of atoms that represents the smallest possible amount of a chemical compound. Molecules can be made of single types of atoms or of different types. For example, the oxygen in the air is made of two oxygen atoms (O2), but water is made of two hydrogen atoms and one oxygen atom (H2O).
nano A prefix indicating a billionth. In the metric system of measurements, it’s often used as an abbreviation to refer to objects that are a billionth of a meter (nanometer) long or in diameter.
National Institute of General Medical Sciences One of the 21 separate National Institutes of Health. This one both conducts internal research and finances research by others into basic biological processes and that may lead to better disease diagnosis, treatment and prevention.
nerve cell or neuron Any of the impulse-conducting cells that make up the brain, spinal column and nervous system. These specialized cells transmit information to other neurons in the form of electrical signals.
optical Having to do with vision or sight, with the fields of optics, or with visible light.
Parkinson’s disease A disease of the brain and nervous system that causes tremors and affects movement, memory and mood.
physical chemistry The area of chemistry that uses the techniques and theories of physics to study chemical systems. A scientist who works in that field is known as a physical chemist.
physicist A scientist who studies the nature and properties of matter and energy.
proteins Compounds made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. The hemoglobin in blood and the antibodies that attempt to fight infections are among the better known, stand-alone proteins.Medicines frequently work by latching onto proteins.
radiation Energy, emitted by a source, that travels through space in wavesor as moving subatomic particles. Examples include visible light, infrared energy and microwaves.
red blood cells Colored red by hemoglobin, these cells move oxygen from the lungs to all tissues of the body.
wavelength The distance between one peak and the next in a series of waves, or the distance between one trough and the next. Visible light — which, like all electromagnetic radiation, travels in waves — includes wavelengths between about 380 nanometers (violet) and about 740 nanometers (red). Radiation with wavelengths shorter than visible light includes gamma rays, X-rays and ultraviolet light. Longer-wavelength radiation includes infrared light, microwaves and radio waves.