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Bio-Rad Microscience Division 19 Blackstone St.Cambridge, Mass. 02139 (800) 444-1422 Fax: (617) 864-9328 Leica Inc. 111 Deer Lake Rd. Deerfield, Ill. 60015 (800) 248-0123 Fax: (708) 405-0147 Meridian Instruments Inc. 2310 Science Pkwy. Okemos, Mich. 48864 (800) 247-8084 Fax: (517) 349-5967 Molecular Probes Inc. 4849 Pitchford Ave. Eugene, Ore. 97402 (503) 465-8300 Fax: (503) 344-6504 Molecular Dynamics Inc. 880 E. Arques Ave. Sunnyvale, Calif. 94086 (800) 333-5703 Fax: (408) 773-8343 Nikon Inc. 1300 Walt Whitman Rd. Melville, N.Y. 11747 (516) 547-8570 Fax: (516) 547-0307 Noran Instruments Inc. 2551 W. Beltline Hwy. Middleton, Wis. 53562 (608) 831-3883 Fax: (608) 836-7224 Olympus Corp. 4 Nevada Dr. Lake Success, N.Y. 11042 (800) 446-5767 Carl Zeiss Inc. One Zeiss Dr. Thornwood, N.Y. 10594 (800) 233-2343 Fax: (914) 681-7446 Recent improvements to the laser-scanning confocal microscope are enhancing the instrument's already impressive capabilities to create sharply defined biological images, researchers say. Advanced techniques, new lasers with added spectral lines, a wider array of sensitive fluorescent dyes, and better image collection and analysis are combining to broaden the powers of these sophisticated instruments. Especially for scientists who image living cells--neurobiologists investigating the actions of neurons, for example--these advances dramatically extend the confocal microscope's usefulness, which has grown steadily since the introduction of the device into labs in the mid-1980s. "Confocal microscopy is certainly the best way we've got of imaging living cells at the limits of optical resolution today," says Stephen J. Smith, a neurobiologist at the Stanford University School of Medicine. According to Smith, the confocal microscope is at its best in imaging living cells when used to look into a "thick, rather turbid" specimen, like brain tissue. "Brain tissue is translucent," Smith says. "So, once it gets more than a few cell layers thick, you can't see through it. Light goes through it, but it's diffused. That's the kind of situation where the confocal microscope comes into its own." The unique advantage of the confocal microscope is its ability to reject this out-of-focus light, or flare, from areas within the specimen other than in the plane of focus. To do this, the microscope first focuses an illuminating cone of laser light through a stack of lenses to a point at a predetermined depth within the specimen. This point is then system- atically scanned back and forth under computer control. In this way, the entire plane of focus is illuminated, but sequentially, point by point, rather than simultaneously, as with an ordinary microscope. The imaging light returning from the specimen, through the same lenses and scanners as before, is separated from the illumination light by a beam splitter. It then passes through a pinhole aperture, called a spatial filter. It is this spatial filter that defines the confocal microscope. The filter has the effect of screening out light from all but the focal point. The remaining, in-focus light is registered with a photomultiplier tube and, with computer assistance, is used to construct a strikingly clear and precise two-dimensional image, called an optical slice or section. Multiple sections can be used to build three-dimensional images, even three-dimensional moving images. "The trick is to get rid of all the illumination in the sample except right at the little point of focus, get rid of the double cone of light above and below," explains Watt W. Webb, a biological physicist and a professor of applied physics at Cornell University. "That's what the aperture does." Most commonly, confocal microscopes are used with fluorescent dyes. When attached to monoclonal antibodies, these dyes have the great advantage of being able to label specific molecules for imaging. When thick specimens are stained and viewed with ordinary microscopes, fluorescent light from the out-of-focus planes can obscure the image. The confocal microscope, viewing the same specimen, is able to eliminate this out-of-focus light to produce sharp images. The confocal microscope's ability to clearly image living cells, in tissue or in culture, will become increasingly important in the near future, according to James B. Pawley, a professor of zoology at the University of Wisconsin, Madison, and editor of a standard reference work in the field (Handbook of Biological Confocal Microscopy, New York, Plenum Publishing Corp., 1990). "The most rapidly developing area is going to be living-cell confocal microscopy," Pawley says. "That's the wave of the future. Of course, it's difficult, because you have to worry about keeping the light dose to an absolute minimum so the cell is not upset." Stephen W. Paddock, a cell biologist also at the University of Wisconsin, Madison, makes the same point: "The problem with imaging living cells is that they are very light-sensitive." Important to the confocal microscope's capabilities is its use of a laser light source to brilliantly illuminate the specimen. While there are many advantages to using such a bright light source, researchers find they must take steps to avoid the injury to cells that can result. "Lasers are just so convenient," says Pawley, "because they are so intense. But one of the problems in the early days of confocal microscopy was that people would turn up the knobs to get a picture quickly, and then they'd look and say, `It bleached so fast!' " Cornell's Webb and graduate student David Sandison have worked out a number of tactics that obviate the problem of delivering too much light to living cells under study. Webb explains that the final image obtained from a confocal microscope is sharper when the spatial filter aperture is smaller, but that more potentially damaging light must then be used to produce an image. "If you make the pinhole spatial filter small enough with confocal microscopy, you improve the resolution of the microscope, even in the plane of focus," Webb says. "But that's not a good idea if you're looking at a biological specimen, because it uses so much light that you overilluminate the specimen. And that's where the damage comes from." People have been tempted to take advantage of the theoretical improvement in resolution of confocal microscopy compared to full-field microscopy, Webb says, by using more intense light and a smaller aperture. In doing fluorescence microscopy, which is what most of the living cell work is, Webb says, this is the wrong direction to go. "Instead, you just open your pinhole a little bit, and take advantage of the removal of flare," he says. "You still come out ahead." A degree of judgment is called for, in other words. The relationship between these two--the size of the aperture opening and the amount of light used--must be optimized to view living cells. Webb says his group has worked out these relationships in "sickening mathematical detail," and that there needn't be excessive damage to the specimen. The overall amount of light used to illuminate a cell is only one way to consider the light energy reaching the cell. Another is to realize that the lasers used in confocal microscopy emit light in different, specific wavelengths, each with its own energy level. A laser commonly used in commercial confocal microscopes, for example, is the argon laser, which emits two major spectral lines, at wavelengths of 488 nanometers (blue) and 514 nm (green). Developers of fluorescent dyes--or reporter molecules-- look for those that are easily excited by these wavelengths and will therefore provide the most information. Recently, confocal microscopes based on a krypton-argon laser that emits three lines, at 488 nm (blue), 568 nm (yellow), and 647 nm (red), have been introduced. The three spectral lines offer a different set of wavelengths for developers of fluorescent dyes to work with, as well as the opportunity to image three separately tagged molecules at once. In addition, the longer-wavelength red line is less damaging to cells under study, according to Paddock. "If you use a shorter wavelength, which has more energy," he says, "it tends to kind of fry the cells more quickly than using a longer wavelength." Paddock, with colleague Steve Stricker at the University of New Mexico, Albuquerque, has used calcium-sensitive fluorescent dyes to image the fertilization process within sea urchin and starfish eggs. "It's long been known that during fertilization, there's a wave of free calcium that passes across the egg," Paddock says. "Using confocal microscopy, you can actually image the wave passing across the egg. We found that the maternal nucleus lights up, as well, which you can only see using confocal microscopy." Roger Tsien, a professor of pharmacology and chemistry at the University of California, San Diego, is well known for having constructed a number of fluorescent reporter molecules sensitive to, among other substances, calcium and cyclic AMP. In addition, he more recently has developed techniques to produce images much faster than is possible using commercially available systems. "The commercial systems typically produce images at about one or two per second, which is too slow to work on many phenomena in living cells," Tsien says. Tsien says his group has been able to build a confocal microscope capable of video-rate imaging--30 frames a second--with full laser scanning. "In the calcium area, we've been looking mainly at excitable cells," he says, "things like heart muscle or neurons, which have fast calcium spikes or calcium waves passing through them. In the cyclic AMP area, we've been looking at the famous model of invertebrate plasticity, the sea slug. This is a long-established model, involving neurotransmitters raising cyclic AMP, which affects synapses for other neurons. We've been looking at how the cyclic AMP changes, which is visible now, for the first time, directly." microscopy to create images of living cells showing cellular activities, using dyes and video techniques such as those he and his group have developed, will lead to important changes in cellular research. Traditional biochemistry, he says, involves "grinding up a couple million cells and running them out on a gel, or doing PCR [polymerase chain reaction] on a whole population," which is useful for ascertaining certain general attributes of that population, but obscures the activities of individual cells. "When you can see their individual biochemical signals," he says, "you find that different cells are often very individualistic, almost like wild animals, or people. "Even different parts of the cell have different biochemistry. The cell is not just a featureless bag. It knows which direction it's headed, so its front and its back have to be different, biochemically." Tsien adds: "Overall, that's where all this live cell work is needed."

Confocal Microscopy: Viewing Cells As """"Wild Animals""""

Jan 24, 1993

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