Innovative instruments, often jerry-built from parts of other devices, are making a wide array of new projects possible
Confocal microscopes have been transforming the working lives of scientists like Sunderland ever since the devices began to appear in biology labs in the mid-1980s. And it's not just their pictures that are causing scientists to want their own. It's also the challenge of improving on the clunky first generation of confocal microscopes.
From the start, confocal microscopes have attracted all sorts of tinkerers and handymen, including Sunderland and inventor Jeff Lichtman. "It was Scotch-tape science," says Sunderland, now pursuing a Ph.D. in molecular biology at the University of Washington in Seattle. "We actually just made the first [microscope] from pieces hanging around the lab. It took about a day."
Sunderland, an undergraduate at Washington University in St. Louis in 1985, had been hired by a cell biology lab to tinker with the computer imaging programs attached to a prototype confocal microscope. The microscope, which has a unique ability to manipulate light, is a complicated assembly of mirrors and lenses designed by Lichtman, the lab's neurobiologist. It was one of a number of different confocal microscopes being developed around the world at the time. For Sunderland it was, to say the least, an eye-opening experience. Snake neurons magnified and projected onto the microscope's video screen were far more detailed than anything he had seen before. The image could be seen not only on a video screen, but also through an eye piece.
The confocal microscope has motivated biologists across the country to return to old scientific projects abandoned because of the limitations of their previous microscopes. The new microscopes are powerful enough to give biologists previously unattainable views of cells, and yet they are still crude enough to inspire laboratory tinkerers to improve their technology. These cell biologists, pathologists, neurologists, and endocrinologists have persuaded the National Institutes of Health to shell out millions of dollars this year to buy the machines, which cost anywhere from $20,000 to $300,000. And over the past two years, a half-dozen companies have come out with their own versions of the confocal microscope (see accompanying story).
What makes the confocal microscope different from other microscopes is how it manipulates light. Only tiny regions of the specimen are illuminated at any one time. In traditional microscopy, light reflected from other parts of the sample tends to cause haziness. The configuration of the confocal microscope eliminates that. A tiny shaft of light first bounces off the specimen and then passes through a pinhole on its journey back to the viewer's eye. That helps screen out excess light. Though some confocal microscopes take advantage of the intensity and coherence of laser light, other confocal microscopes use ordinary white light, which is cheaper.
Confocal microscopes are magic wands in laboratory science, say their users. "There is a sort of witchcraft" in using his microscope, says one inventor of a confocal microscope, physicist Lars Majlof, who is also vice-president of Sarastro, a five-year-old Swedish firm that has sold 10 of his confocal microscopes (each at $175,000 or more). And the results are a bit unbelievable. "There is the feeling of seeing something supernatural," says Majlof, "because in the image that appears you see things that are not there when you look into an ordinary microscope."
| Confocal microscopes are a boon to cell biologists. They've also found a ready market in the materials, pharmaceuticals, and electronics industry. Leica sells a scope to pharmaceutical companies that analyzes the roughness of pill surfaces. A microscope manufactured by Tracor-Northern is used to detect asbestos fibers in school floor tiles. A Technical Instrument microscope burrows down through the layers of a semiconductor chip in search of defects. Following are some of the companies that sell confocal microscopes for these and other uses: |
Technical Instrument Co.
Meridian Instruments Inc.
Leica (Wild Leitz USA Inc.)
Carl Zeiss Inc.
For an essential guide and extensive bibliography to the science of confocal microscopes: Handbook of Biological Confocal Microscopy, edited by James B. Pawley. Plenum Press, New York, 1990.
"If you're working with a thick cell, with a conventional light microscope, you get intensity from all different focal planes, and a hazy background," explains Fred Maxfield, a cell biologist at Columbia University in New York. "The confocal rejects out-of-focus light, so you get sharp images of structures. Suddenly you can actually see the cytoskeleton." Maxfield says the laser confocal will allow him to revisit projects put aside a couple of years ago, such as one looking at how polarized cells transport proteins tagged with a fluorescent dye.
The principle of shooting thin points of light at a sample is simple and dates to the 1950s. But it wasn't until the 1970s, when biologists began to use fluorescent tags, that the idea was recognized as a useful tool. Scientists hoped the microscope would solve the main problem with fluorescent dyes: that they tend to scatter light, making images hard to study.
"Work on confocals and fluorescence converged in the late 1980s," says neurobiologist Lichtman. Next came the use of the intense and convergent light of a laser, which gave cell biologists an even more powerful tool. Laser microscopy was made possible by advances in computer programming known as digital image processing.
The following step was to produce a three-dimensional view of a sample. To do this, the microscope scans a specimen. It then scans at a slightly deeper plane, say, a half-micron beneath the previous level, repeating this 30 to 50 more times. Each image is converted into a set of numbers. The microscope's computer program then compiles a host of these images--each image taken at a slightly different depth. The crisp picture on the video screen appears to be a three-dimensional image of the cell. An image processor manipulates the numbers that represent the image to display the cell from a different angle.
"It lets you see relationships [between cell components] that were hard to see with stacks of two-dimensional images," says Maxfield. "You get a sense of the three-dimensional architecture of the cell. [For the first time] I can see the organelle's almost fingerlike projections."
This detail can have a direct effect on the progress of a scientist's work. "The advances we've made [using the microscope] over the last three months would probably have taken us three years," says cell biologist James Truman of the University of Washington. The microscope allows him to track the movement of antigens in the immune systems of insects. "With the confocal it's fairly easy to see how things relate spatially," Truman explains. "In the past you had a whole pile of separate files. You would have to reconstruct the [insect] systems on paper, and we couldn't do as thorough an analysis."
As excited as they are by what the confocal allows them to see, some scientists are having fun just making and improving the devices. Their goal is to take the witchcraft out of the science.
"With the first laser microscopes, you needed to twiddle with the electronics to get the optimal images," says Washington University's Lichtman, one of the grand tinkerers of confocal microscopes. Whether a scientist could get them to work seemed to depend on equal amounts of witchery and ingenuity. But the newer models still have their share of shortcomings, especially for scientists who use powerful lasers to look at living cells.
"With [a 1987 version of the machine made by Bio-Rad] things tend to get fried," says Truman. For cell biologist David Bentley, at the University of California in Berkeley, it meant giving up the use of confocals altogether. He says that when the fluorescent tags in his samples were stimulated by the laser light, the energy generated was so intense it damaged the living insect nerve cells.
Now companies--and the scientists themselves--are experimenting with ways to reduce the amount of light needed. They see these current microscopes as the Model Ts of this technology, requiring a scientist to be mechanically minded to make the best use of them.
Steve Smith, a neurobiologist at Stanford University, takes the garage mechanic's approach to confocal microscopes. With the help of Tim Ryan, a physicist who works in his lab, and a lot of electronic "twiddling," Smith is now able to use low light with his first-generation Bio-Rad to make movies of living brain cells.
"I played around with the engineering to allow [the microscope] to collect the most possible photons from the specimen, which allows us to put the fewest possible excitation photons into the specimen," Smith says. "It took about five minutes to figure out. [Now the microscope] has a little knob the user has to adjust every time he turns on the machine."
This need for constant adjusting is one drawback of confocal microscopes. (Bio-Rad believes its new generation microscope takes care of the problems with laser intensity.) A second drawback is the price tags. To afford the machines, many scientists say they must share them with other labs. This year 11 institutions are receiving more than $2 million to buy the microscopes from the National Institutes of Health through its Shared Instrumentation Grant program.
A half-dozen companies are putting out confocal microscopes, but scientists themselves are also trying to make the devices more user-friendly and less costly. After a lot of fine tuning, Smith's colleague Lichtman is planning to sell his white light microscope to other biologists this fall through the Newport Corp. in Fountain Valley, Calif. At $20,000 this "poor man's" microscope is certainly more affordable than the $120,000-to-$300,000 price tag for a laser microscope from Bio-Rad of Cambridge, Mass., Sarastro of Stockholm, or Leica of Rockleigh, N.J.
The more expensive laser machine is essential for work with fluorescent dyes. But the white light microscope is more affordable.
"The biggest problem with [confocal microscopes] is not that you need to hold a wrench to get the best results. It's the expense," says Smith. "There are thousands of people in laboratories who want one and should have one," but can't afford them. However, Smith expects some biologist-entrepreneur like himself to refine this technology until, one day, every lab will have a laser confocal microscope of its own.