Science/Medicine : The X-Ray Holograph

This remarkable new imaging technique is being developed to enable biologists to view all but the smallest components of living cells.

Humans have long had a fascination with things too small to be seen with the naked eye.

As long as 3,000 years ago, engravers used glass globes filled with water as magnifiers as they worked so that they could see fine details of their designs. Ancient Romans used crude lenses chipped from rock crystals for the same purpose.

Interest in things small was strengthened in the mid-1600s when Dutch amateur scientist Anton van Leeuwenhoek used a simple microscope of his own design to see, for the first time, bacteria and other tiny living organisms.

Today researchers are developing a remarkable new imaging technique called X-ray holography that they believe will enable biologists to view all but the smallest components of living cells much more clearly than is now possible and in three dimensions rather than two, experts say.

The new technique may thus provide new insights into how cells and their components are constructed, according to physicist Malcolm Howells of Lawrence Berkeley Laboratory. Such insights, in turn, may lead to new understanding of diseases and new treatments.

Perhaps more important, he said, X-ray holography may allow for the viewing of movements and interactions of subcellular structures while the cell is still alive--a feat not now possible.

Cystic fibrosis, for example, is thought to involve a defect in the way cells lining the lungs secrete mucus, said physiologist Steve Rothman of Berkeley. If researchers were able to view the cells during the secretion process by X-ray holography, he said, “we might learn a lot more about how the disease works.”

Light microscopes--which use glass lenses at opposite ends of a short tube to magnify objects--have been the mainstay of microbiologists since Dutch spectacle maker Zacharias Janssen invented the first one in 1590.

But such microscopes cannot be used to view anything smaller than the wavelength of light--about 8 millionths of an inch. Many components of the cell are thus too small to be seen.

Objects too small to be seen in a light microscope can be seen with an electron microscope because the wavelength of electrons is much shorter than that of light. The electron microscope--which was developed in 1933, but not applied extensively to biology until the 1950s--works just like a light microscope, but a beam of electrons is used instead of light and the beam is focused with magnets rather than lenses.

But a specimen to be viewed in an electron microscope must typically be embedded in a plastic matrix, stained to enhance contrast, and cut into very thin slices so that an electron beam can pass through it. This process stops biological activity and can distort the specimen’s structure.

Like electrons, X-rays have a short wavelength and could be used to view extremely small objects. They also have a greater penetrating power than electrons, so that slicing the specimen would not be necessary. But X-rays cannot be focused by lenses; X-ray microscopy thus requires the use of holography.

Holography, a technique for producing three-dimensional images, was developed in 1949 by Hungarian physicist Dennis Gabor, who won the 1971 Nobel prize in physics for the feat. The technique can be used with any form of energy that is carried by waves, including sound, light, microwaves and X-rays, but the most work has been done with light.

In conventional photography, light reflected from a subject is focused on film with a lens. When the film is developed, a flat image is seen.

But a hologram is recorded without lenses, using a table top-sized apparatus for small subjects--say a few inches tall--and a room full of equipment for larger objects.

To record a hologram, a laser beam is split with a prism. One half of the beam strikes the film directly, while the other half strikes the object and is reflected onto the film. When the film is developed, no image is seen. Instead, the film looks like it is covered with smudged, wavy lines.

When laser light shines through the film, the viewer sees a three-dimensional image of the original subject--a statue, say--as though it were being viewed through a window. By moving from side to side, the viewer can even see details of the statue that are hidden at other angles, just as though the statue were being viewed directly.

Holographic images are used to reproduce objects of art or as art objects themselves. The images and technology are used in advertising, in scanners at checkout counters in supermarkets and for finding flaws in manufactured goods such as turbine blades and jet engine parts.

Through special processing, holograms can also be imprinted on plastic so they can be viewed with normal light. These images have appeared on two National Geographic covers, on Ghost Busters cereal boxes and packaging for Polaroid’s Onyx camera, on toys such as Hasbro’s Iron Mountain play set and Tonka’s Supernatural figures and on MasterCard and Visa credit cards.

The key to producing holograms with light is the use of a laser. All the light from a laser is of the same wavelength and traveling in the same direction; and all the vibrations are synchronized. Laser light is like a platoon of soldiers marching in lock step. Such “coherent” light produces sharp, clear holograms.

Light from a normal bulb, in contrast, has many different wavelengths, travels in all directions, and the vibrations are not synchronized. It is more like a crowd of pedestrians strolling through a train terminal. Such “incoherent” light can be used to make holograms, but the images are weak and indistinct.

X-rays produced by a normal X-ray tube are incoherent like light from a bulb. They do not make good holograms.

But new sources of coherent X-rays more suitable for holography have become available recently as spinoffs from research on particle accelerators, laser-based fusion for energy production and the Strategic Defense Initiative, according to physicist Nat Ceglio of Lawrence Livermore National Laboratory.

About two years ago, for instance, physicists began to use the undulator, which produces very intense X-rays from the electron beam in a synchrotron. Synchrotrons store beams of electrons produced in a particle accelerator by using magnets to deflect them around a circular pathway.

The undulator uses magnets to bend the beam of electrons from side to side very rapidly so that it undulates like a snake. That motion releases an intense laser-like beam of coherent X-rays. But unlike the X-rays used in medicine, these have a very low energy: they will not even pass through a sheet of paper.

Howells and his colleagues at Berkeley have used the undulator at the Brookhaven National Laboratory’s synchrotron on Long Island, N.Y., to record holograms of zymogen granules--roughly spherical sacs from the pancreas that contain digestive enzymes to be released into the stomach. These granules are just the right size for viewing by X-ray holography, Rothman said.

The hologram obtained in this way is too small for an image to be reproduced with visible light. The Berkeley group thus takes a picture of the hologram through an electron microscope and converts that photograph into numerical data in the same way that satellite pictures are converted into digital signals for transmission to earth. A computer then processes the data to produce an image.

The chief advantage of X-ray holography is that “it doesn’t require extensive sample preparation,” Rothman said. When zymogen granules are processed and viewed in an electron microscope, he said, they appear to be uniform throughout their interiors, like a rubber ball that has been sliced open.

But the X-ray holographic image of unprocessed granules shows structure inside them, he said, and a knowledge of that structure may help explain how the granules function.

X-ray holograms of microscopic fibers have been produced at Livermore using an X-ray laser developed by physicist Dennis L. Matthews and his colleagues. It is powered by two of the 10 massive lasers in Livermore’s football field-sized Nova Laser Facility, which is used for experiments in using laser-induced nuclear fusion to produce electricity. The two lasers are focused on a 2-inch-long selenium fiber much thinner than a human hair.

When the two lasers are fired simultaneously, the selenium explodes, producing a hot gas that emits a pulse of coherent X-rays about 200 trillionths of a second long and directed where the thread was pointed.

An X-ray laser being developed at Livermore for the Strategic Defense Initiative uses a similar principle, but it would be powered by a nuclear explosion and could destroy rockets launched in an attack against the United States.

Physicist Szymon Suckewer of Princeton University has made a smaller X-ray laser that he said is at least 100 times more efficient than the Livermore laser, and thus does not require such massive lasers to power it. He is now attempting to make holograms with it.

Because of the laser’s efficiency, he said, it could be fired about every two minutes to make a series of holograms of a living cell--although other researchers say such repetitive firing would destroy the specimen. The power supply of the Livermore laser, in contrast, must be charged for several hours before it can be fired.

The few holograms produced at Berkeley and Livermore so far are crude and “nothing earthshaking in themselves,” said physicist Herman Winick of Stanford University, but they show that X-ray holography works. “We are really excited about the future potential of the technique,” he said.