Improvements In Light Microscopy Lead To New Applications

Hosts of new applications have turned the light microscope into an exquisitely sensitive measuring device that can be used to image dynamic cellular events right down to the molecular level. Indeed, dramatic improvements in optical microscopy have brought about a renaissance in the use of light microscopes in biology and medicine. And within the last few years, the combination of novel optical systems with video and digital image processing capabilities have opened up whole new fields of research in biology, chemistry, and even physics. The invention that first enabled researchers to see clear images of living cells was the phase-contrast microscope, which won its inventor, Frits Zernike, a Nobel Prize in 1932. Prior to Zernike's work, specimens were typically stained with organic dyes with such colorful names as coomassie blue and malachite green. These dyes either stained the whole specimen to provide contrast or, preferably, stained only parts of cells, allowing researchers to look at specific cellular components like the nucleus and cell membranes. Fixing and staining specimens, however, killed the cells and distorted their appearance. Following Zernike's invention, another contrast-generating microscope, the differential interference-contrast microscope, was invented by G. Nomarski in 1952. In the ensuing four decades, contrast microscopes have become standard equipment in cell biology laboratories. "The best way to look at living cells today is using phase- contrast or differential interference- contrast microscopy," says neurobiologist Christopher Case of the State University of New York, Albany. Contrast-generating techniques require the addition of special systems to the light microscope that exploit changes in the phase of light waves as they pass through different parts of cells. The high-contrast images obtained from these microscopes help researchers like Case examine the location and function of such cellular structures as neurons and mitochondria. Studies of the nematode Caenorhabditis elegans, begun in the 1960s by Sydney Brenner at the Medical Research Council in Cambridge, England, depended to a large extent on Nomarski optics, which allowed researchers to see--in exquisite detail--cell division during development of the transparent worm (The Scientist, Oct. 28, 1991, page 22). An important technique to grow out of light microscopy is fluorescence microscopy, which enables the imaging of very weak light signals emanating from luminescent molecules. The optical system in fluorescence microscopy is similar to basic light optics, except that the detection system is modified so that only certain wavelengths of light--those that have been emitted as higher-wavelength (lower-energy) light from excited molecules--are detected and used to create the image. The development of epifluorescent illumination (in which the specimen is lighted from above) in the mid-1970s is viewed by many researchers as one of the most important recent improvements in light microscopy. "In the past 10 to 12 years there has been a revolution in microscopy because of the development of techniques like epifluorescence, especially with fluorescent tags," says research scientist Jeff Travis of SUNY-Albany's department of biology. Epifluorescence uses a single objective lens and a dichromatic beam splitter that filters out any wavelengths of light that would make the image appear out of focus. Using fluorescence microscopy in conjunction with the plethora of fluorescent probes now available makes this technique a very powerful tool for imaging cells or parts of cells. And combining these techniques with spectroscopic measurements has extended the power of fluorescence microscopy to the molecular level. For example, some of the fluorescein dyes used as probes display shifts in their emission spectrum in response to chemical changes in their environment. The pH or calcium ion concentration found in a particular region of a cell can be quantified by measurement of these shifts over time. Other fluorescent compounds selectively enter various cellular components, thus providing vital staining of specific organelles. Combining the specificity of antibodies with fluorescence in immunofluorescent probes allows the detection of target molecules both inside of a cell and out. By microscopically observing the pattern of fluorescence in a specimen that has been dyed with immunospecific fluorescent dyes, a researcher can determine the exact location of specific cellular proteins. Many investigators, including SUNY's Case, use a combination of phase-contrast or differential interference-contrast microscopy and fluorescence microscopy to first create high-contrast images of structures within cells, and then stain these structures with specific immunofluorescent probes and look at them again. By far the most exciting recent advance in fluorescent microscopy came with the development of confocal microscopes, widely regarded to be the impetus behind the recent explosion in microscopy research. "Confocal microscopy is a different way of imaging," says Jim Turner, a research scientist at the Wadsworth Center for Laboratories and Research in the New York State Department of Health. "You get vastly improved resolution and three-dimensional images," as opposed to the generally two-dimensional images created by the light microscopes. "Just a few years ago, thick objects could not be imaged with a light microscope. Now we can look at objects half a millimeter thick, like large cells in situ," says Turner. "It's fantastic what we can do." What differentiates confocal microscopes from other microscopes is an aperture the size of a pinhole located in front of the photodetector, which blocks the transmittance of out-of-focus light (The Scientist, July 23, 1990, page 1). In conventional light microscopy, incident light reflected from other parts of a specimen creates an out-of-focus "haze." The confocal microscope is set up so that a single point of light beams down to the specimen and then bounces back up through the pinpoint aperture on its way to the viewer's eye. The design of the device effectively screens the out-of-focus light, and eliminates the blurry background haze. The idea of illuminating specimens with narrow beams of light has been around since the 1950s. Early versions of the confocal microscopy used ordinary white light. With the advent of laser technology, researchers found that the power of the technique could be increased by combining confocal microscopy and the intense, cohesive light of a laser. The recent convergence of laser-confocal microscopy and fluorescence has many researchers heading back into the laboratory to look at cells in different ways. David Ward and his colleagues at the Yale University School of Medicine are using fluorescent-labeled DNA probes to locate specific genes on human chromosomes, an application that many find important as the journey through the human genome progresses. Development of this technique, which involves both conventional and laser-scanning confocal microscopy, gives gene mappers a place to start in their search for specific genes. By providing the approximate location of a gene on a particular chromosome, this technique may speed the expedition through the 3 billion pairs of DNA bases in the human genome. Jim Hayden, product specialist for Bio-Rad Laboratories' Microscience Division in Cambridge, Mass., explains how laser-scanning confocal microscopy works: When laser light strikes the surface of a specimen, only a single point is illuminated. "The laser light strikes a [fluorescent] specimen and it excites the dye, which then releases longer wavelength light," Hayden says. These "points" of lower-energy light are detected by the photodetector and then sent to a computer for imaging. "It starts with one point," he says, "and then the computer builds up points over a layer, like a TV that scans over the screen." Since the in-focus plane alone is transmitted, only a very thin slice of the specimen is imaged. Images from various planes in the specimen can be digitized by a computer and then compiled, forming a reconstructed, three-dimensional image with "greater resolution and less out-of-focus information," according to Hayden. "We can see things clearly that we could never have seen before," says Wadsworth's Turner. A recent improvement in laser-confocal technology by researchers at Cornell University, called two-photon excitation laser-scanning microscopy, uses extraordinarily brief pulses of red laser light to excite the fluorescent dye. Since the dye is excited only at the focal point of the laser beam, very little photobleaching or fluorescent background occurs. Some of the most important developments in microscopy have resulted from the successful marriage of microscopes to video electronics and computers. "Vast improvements have come with the application of video and digital image processing," says SUNY-Albany's Travis. Linking a microscope to a video screen allows a researcher to project images of a particular microscopic specimen onto the screen and record what happens in sequence, instead of sitting for hours peering through the ocular lenses. This technique also allows time-lapse photography of cells undergoing mitosis and other functions. The addition of a computer to the system allows the user to do things the eye or a camera alone cannot. In computerized image processing, the video camera captures and records images that are beyond the viewer's capability to detect. The computer then amplifies the signal. This translates to improved detectability and resolution. The video image can be digitized by the computer as an array of numerical data points, which can be manipulated in all sorts of mathematical ways. Researchers can even separate the important information away from the background noise. "A lot of people trained in science don't think of images as data," says Travis. "With the computer, you can take the data and then highlight the parts of the image you want, and eliminate the parts you don't want." Improved contrast and less background are the net result. This is especially important to users of fluorescence microscopy, because the low-intensity light emitted from the excited fluorochrome is not always detectable without video enhancement. When two different fluorescent labels are used in an experiment, the distribution of one can be examined while blocking out the other, and vice versa. Recent refinements and applications that just seem to keep coming have created a revolution on all fronts of microscopy. "It's an exciting time to be in the field," says Turner. Adds Travis, "It's like it's okay to go look at living cells in a microscope again, because now you can see subcellular features." Holly Ahern teaches cellular and molecular biology at the State University of New York, Albany.

Improvements In Light Microscopy Lead To New Applications

Interested in reading more?

Become a Member of