Technology Advances Marking Milestones In Microscopy

Microscopy Sidebar: Selected Vendors Microscopy makes a big deal out of little things. And one of the biggest things to come out of microscopy this year was evidence of life on Mars. Using new tools in high-resolution scanning electron microscopy, researchers peered at tiny structures found deep within fractures of a 4.2 pound, potato-sized meteorite and concluded they had found evidence of "primitive life on early Mars" (D.S. McKay et al., Science, 273:924-30, 1996). But alien microfossils aren't the only news in microscopy. Innovations in technology and technique mean bigger deals can be made out of ever smaller things. Several dozen microscopy specialists responded to E-mail and phone queries from The Scientist with opinions about the most notable new technologies. "One test of all the exciting things out there is to make yourself a wish list and then go to the floor of our trade show, which is the principal forum of exchange for new information about microscopes and microanalysis," says Ann Goldstein, president of the Pocasset, Mass.-based Microscopy Society of America (MSA). "You'd be surprised at how many things that you thought would be nice are already there or are in testing labs and on their way to the market." Advances in electron and atomic force microscopy provide some of the highest-resolution images of life's tiniest dings, nicks, and striations. In electron microscopy, exceptionally bright and narrow electron sources known as field emitters or field emission guns have been a major advance, according to Nestor Zaluzec, a staff scientist in the Materials Science Division of the Argonne National Laboratory in Illinois. Zaluzec was program chairman for Microscopy and Microanalysis '96, the joint annual meeting of MSA, the Microbeam Analysis Society, and the Microscopical Society of Canada, held in Minneapolis in August. Supplies AMRAY Inc. Applied Precision Inc. AutoQuant Imaging Inc. Bio-Rad Laboratories Inc. Chroma Technology Digital Instruments Inc. ElectroScan Corp. Hitachi Instruments Inc. JEOL USA Inc. Leica Inc. LEO Electron Microscopy Inc. Life Science Research Ltd. Meridian Instruments Inc. Molecular Dynamics Inc. Molecular Probes Inc. Nikon Inc. Instrument Group Olympus America Inc. Philips Electron Optics Princeton Instruments Inc. Topcon Technologies Inc. Vysis Inc. Carl Zeiss Inc. A limiting factor in the resolution of images from a scanning electron microscope is the size of the electron beam that hits the sample. The bright, intense new electron source known as the field emitter has increased resolution to less than 1 nanometer in diameter. That's small enough to show that cell adhesion molecules on the surface of leukocytes are focused at the tips of the cells, a mechanism important to the body's injury response, explains Stanley Erlandsen, an electron microscopist in the cell biology and neuroanatomy department at the University of Minnesota Medical School in Minneapolis. Erlandsen uses a Hitachi S-900 field emission SEM, made by Hitachi Instruments Inc. of San Jose, Calif. It offers a very short focal length, hence the highest resolution possible, he says. Another recent advance has been the low-vacuum scanning electron microscope, enabling researchers to look at moist biological samples, like a tissue biopsy or a leaf, without first drying or dehydrating them, reports Bob Santorelli, vice president and general manager of the electron optics division of JEOL USA Inc. The Peabody, Mass.-based subsidiary of Japan Electron Optics Laboratory Ltd. offers several low-vacuum scanning models, including the JSM-5800LV. Another electron microscope maker, AMRAY Inc. of Bedford, Mass., offers an environmental-control, variable-pressure model called the 3200-ECO-SEM. In March 1996, ElectroScan Corp. of Willington, Mass., and Philips Electron Optics of Eindhoven, the Netherlands, introduced the first low-vacuum, or "environmental," scanning electron microscope with a field-emission gun. It is marketed as the ESEM 3030FE in the United States, and as the XL30 ESEM-FEG in the rest of the world by Philips, which recently purchased ElectroScan. Other U.S. suppliers of field-emission and variable-pressure scanning electron microscopes include LEO Electron Microscopy Inc. of Thornwood, N.Y., and Topcon Technologies Inc. of Paramus, N.J. These new electron tools mean biological samples have to undergo less preparation, a process that can introduce artifacts that may be hard to distinguish from the data being sought. A drawback to working at the molecular and atomic levels is the slow, often painstaking process of figuring out if what you found is indeed what you think you found. Yet, while it's more difficult to interpret nanoimages, seeing things for the first time at the atomic level is exciting and rewarding, explains Paul Hansma, a physics professor at the University of California (UC), Santa Barbara. "Atomic-force microscopy combines the high magnification of electron microscopy with the ability to work with fully hydrated samples," says Hansma, who pioneered the technique to view samples in fluid environments. The atomic-force microscope feels the contours of objects by tapping its tip across the surface of specimens, feeling contours and monitoring changes in shape. "We're working now on watching individual protein molecules in action. Others are starting to be able to measure forces between molecules holding biological structures together." Hansma and his coworkers primarily use commercial products specially built for biological applications by Digital Instruments Inc. The Santa Barbara, Calif.-based company licensed the UC patents on which Hansma is the inventor and also helps support his work. COMPACT CONSTRUCTION: Digital Instruments’ MultiMode includes an optical detection head, scanner, and microscope base. Three atomic-force microscopes available from Digital Instruments are designed for use with optical microscopes from other companies. The MultiMode is paired with a low-power stereo microscope; the Dimension comes with an integrated video microscope for opaque samples; and the BioScope is matched with an inverted optical microscope, such as the Axiovert from Carl Zeiss Inc. of Thornwood, N.Y. The combination enables the researcher to view cells in a petri dish, for example, through fluorescent microscopy techniques and then zoom in with atomic force microscopy. Improvements in light microscopy and fluorescent techniques that allow life scientists to peek at the detailed activities of living cells are beginning to spark much interest. Researchers using light microscopy generally agree that confocal laser scanning microscopy boosted the resurgence of interest in the technology. The technique removes out-of-focus haze by passing light through one or more pinholes, leaving only a thin, highly focused plane. The light from many such focused planes can be digitized and stored on a computer to create a three-dimensional image analysis. The trend in confocal and other fluorescent equipment is to provide systems at affordable prices that are automated for easier use in multi-user facilities, says David Carter, applications scientist at Meridian Instruments Inc. The Okemos, Michigan-based company (Tel:517-349-7200) just launched its Meridian/TR series. The six models of workstations represent an array of modular components that can be upgraded from a basic to a fully outfitted model that sports four laser lines, including ultraviolet. The new series complements Meridian's InSight and Ultima series. In November, Olympus America Inc. of Melville, N.Y., introduced its second-generation confocal microscope workstation, the Fluoview. It is a billed as an affordable personal laser-scanning microscope, notes Michael Crump, director of marketing for the precision instrument division. "The real emphasis now is on system-related solutions, where the microscope is at the heart of an imaging workstation, often including a highly sensitive camera and software," Crump adds. In partnership with Life Science Research Ltd. of Cambridge, England, Olympus America offers several imaging and photometric systems for real-time quantitative fluorescence microscopy, such as the Mira Cal and BioFluor systems. ALTERNATIVE APPROACH: Applied Precision’s DeltaVision microscope uses digital image processing rather than the confocal method. Vying with the confocal for popular attention is the deconvolution system, which uses complex computer tricks to enhance image detail. Specifically, deconvolution mathematically incorporates data above and below the focal plane (the stuff you can't get into focus). For example, Applied Precision Inc. of Issaquah, Wash., makes the DeltaVision, a wide-field deconvolution microscope that uses digital image processing as an alternative to the confocal approach to high-resolution 3-D fluorescence microscopy, according to Carl Brown, chief scientist at the company. But a new type of fluorescent microscopy is now hitting the market. "One of the biggest items on the horizon is two-photon microscopy," says Marshall Montrose, a cell physiologist in the department of medicine at Johns Hopkins University. Two-photon microscopy users a powerful infrared laser to excite the fluorescent molecules, which mimics the effects of the shorter-wavelength ultraviolet lasers used by confocal microscopes. The idea is that two long-wavelength (low-energy) protons arrive in such quick succession that they have the impact (energy) of light with half the wavelength. In an important advantage, the two photons will act this way only on a small spot, right at the focal point of the laser within a cell. Where confocal laser imaging excites fluorescent molecules along the entire optical path-"bleaching" away other items of interest from view, or unintentionally exciting fluorophores outside the field of view-two-photon laser imaging targets only the limited focal point in space. Two-photon technology is anticipated to be adept at deep-tissue imaging, uncaging of fluorescent reporter molecules at specific subcellular sites, and other applications requiring pin-point accuracy. Watt Webb's group in applied and engineering physics at Cornell University invented the system in the late 1980s. Several months ago, Cornell's technology transfer office granted exclusive commercialization rights to Bio-Rad Laboratories Inc. of Hercules, Calif. According to Gerhart Kneissl, manager for technical resources, this spring Bio-Rad plans to roll out a dual-laser system that combines two-photon technology and the company's conventional confocal product. Kneissl notes that there are experiments in which a conventional confocal microscope would work better than a two-photon system. If, for example, the sample contains color that absorbs infrared light, such as plant cells containing chlorophyll, then confocal would be the preferred approach. "The last few years have seen a number of exciting developments in light microscopy, including a huge array of new and highly sensitive fluorophores and dyes than can be used to visualize structures, molecules like mRNA, and even processes like changes in calcium concentration or voltage changes in living preparations," says University of Arizona neurobiology researcher Lynne Oland. New offerings from Molecular Probes Inc. of Eugene, Ore., address problems associated with calibration of confocal laser-scanning microscopes, photobleaching, and viable cell tracing, says company technical writer Stephen Chamberlain. FocalCheck microspheres (15 micron polystyrene beads with distinct ring-staining patterns) allow precise calibration of confocal microscopes prior to subjecting valuable tissue specimens to laser emission. To reduce photobleaching, the company's ProLong kit provides both antifade reagent and mounting medium. For tracing viable cells over several days, thiol-reactive CellTracker probes are dyes that are retained in live cells through cell division. A growing field, fluorescence microscopy is increasingly becoming a tool of clinical laboratories as well as a tool of basic research. For example, Vysis Inc., the Downers Grove, Ill., company holding exclusive worldwide licenses for the fluorescence in situ hybridization (FISH) methodology and platform, just announced that it received Food and Drug Administration "clearance" for its CEP 8 SpectrumOrange Direct Label Chromosome Enumeration DNA Probe Kit to be used as an adjunct to standard cytogenetics using nonspecific stains, reports Russell Enns, vice president of regulatory affairs at Vysis. Fluorescence microscopy also depends upon optical filters, coatings on glass or quartz. Some filters purify the incoming light to make a color that the probe absorbs well; others clarify the outgoing image by blocking the original light yet letting through the probe's fluorescence. Filters are typically matched to the dyes, or fluorescent probes. For Paul Millman, vice president of sales and marketing for optical filter maker Chroma Technology Corp. of Brattleboro, Vt., one of the hottest issues of the year is increased use of green fluorescent protein. Increasingly popular because it doesn't poison cells and comes in more colors, the green fluorescent probe has prompted a reintroduction of the fluorescence stereomicroscope by Leica Inc., according to Andrew K. Lee, Leica's manager for business development and planning. This microscope creates a three-dimension effect by directing two beams of light at the sample. Under a stereo- microscope with modern optics, scientists can view a whole, live fruit fly, nematode, or zebrafish. The Leica MZ12, for example, offers a look at live samples with a resolution and magnification not possible a decade ago, when the stereofluorescent microscope was first introduced, Lee says. NEW FROM NIKON: The E-800 microscope features infinity optics; the system offers a wide selection of objective lenses. Nikon Inc. in Mellville, N.Y., recently added infinity optics to its microscopes. Infinity optics allows rays of light from the sample to come through the objective lens in parallel wavelengths, instead of being focused to a point to form the intermediate image on the objective lens. This design allows greater design flexibility to, say, slip other optical accessories elements into the imaging light path. Called Chromatic Aberration Free Infinity 60mm Parfocal Optical System, or CFI60 for short, Nikon's edge is a longer objective, which has higher numerical apertures, longer working distances, and larger field sizes to gather low light and to allow researchers to see more deeply over larger specimen areas, says Stan Schwartz, biomedical instruments manager for Nikon Inc. Instrument Group. "The biggest innovation now is the switch from traditional photography techniques to pure computer image capture and analysis techniques," maintains Ron Petralia, president of the Chesapeake Society for Microscopy and a senior staff fellow in the National Institute on Deafness and Other Communication Disorders. "The first step is the end of darkroom printing, while still relying on traditional image capture on film. The second big step is direct image capture on the light or electron microscope; it still has a few years to go before capture resolution and data acquisition and storage are optimal for most applications." NEW SYSTEM: Carl Zeiss’s Axioplan 2 INTEGRATION: With the Axiophot 2, shown here, and the Axioplan 2, Zeiss combines digital processing and an automated systems approach. Trends in imaging and digital processing are clear, observes Chris Souwand, biomedical microscopy product manager for Carl Zeiss. With two new light microscopy systems this year-Axioplan 2 and Axiophot 2-Zeiss integrates digital processing and an automated systems approach into multifluorescence microscopy. Another component at the cutting edge in digital imaging in the biological sciences is the scientific grade cooled charge-coupled device (CCD) camera. Princeton Instruments Inc. of Trenton, N.J., has just introduced three new CCD detectors for the biological market. A new back-illuminated CCD detector, called the TE/CCD-1000TD, is the optimal detector for green fluorescent protein-based imaging, says Mark Christenson, applications scientist at the company. A new interline CCD, called the TE/CCD-1300, with high resolution and quantum efficiencies, aims to pick up fluorescent probes in the 500-nanometer zone (FITC, Fura-2, and so forth). A Intensified PentaMax-512EFT, a new CCD with high dynamic range, sensitivity, and resolution, is designed for kinetic imaging applications that are very low light, such as calcium imaging with Fura-2, or fluorescent particle tracking or single-molecule fluorescence, Christenson says. IN STEREO: Leica’s MZ12 is a stereofluorescent microscope offering a high-resolution look at live samples. Leica has introduced CCD technology and software to help take 35-mm photomicrographs using film. The inexpensive camera features an illuminated reticule (light guides help frame the picture compared to black lines) and it can attach to any microscopy. "The resolution on 35 mm is greater than what you get digitally," Lee claims. "The need for film is getting to be less and less, but it's not going to go away." NEW FRONTIER: Molecular Dynamics has released a new version of its ImageSpace software, which works with any commerical CLSM system. One of the new frontiers of quantitative microscopy involves the interpretation and analysis of three-dimensional confocal data, according to Steve Nelson, manager of software products and intellectual property for Molecular Dynamics Inc. The Sunnyvale, Calif.-based company recently released a new version of its ImageSpace software to review, render, and analyze confocal data in two and three dimensions, including quantitative colocalization (the presence of two or more fluorescence labels emitting from the same spatial location). ImageSpace now imports data from all commercial confocal systems and works with any commercial CLSM system, Nelson notes. Also moving beyond imaging to analysis, Badri Roysam, an associate professor of electrical, computer, and systems engineering at Rensselaer Polytechnic Institute, has figured out a way to stitch together analysis of those images. The automated analysis changes the scale of tissue studies from a statistical sampling to a direct quantitative evaluation of a wide area of tissue. Roysam hopes to offer the new software through his company, AutoQuant Imaging Inc. Based in Troy, N.Y., the firm sells an advanced deconvolution software called AutoDeblur. In transmission electron microscopes (for looking through materials compared to a scanning electron microscope that looks at the surface of materials), Hitachi's new H-7500 TEM may be the first such instrument to move to a personal computer. Running on Microsoft Corp.'s Windows 95, the system reduces complex microscope procedures to a few mouse clicks. Soon, the digitized information will make remote microscopy more commonplace through the Internet, satellites, or other communication technologies, predicts Peter Ingram, a senior physicist at Research Triangle Institute in Research Triangle Park, N.C., and an adjunct professor of pathology at Duke University Medical Center. Sums up Jay Jerome, an associate professor of pathology and director of the microscopy resource at Bowman Gray School of Medicine of Wake Forest University: "Change is not coming as major advances; rather, each year our ability to view and analyze images becomes strengthened by incremental changes." Carol Cruzan Morton is a freelance science journalist in El Cerrito, Calif., and a science writer at University of California, Davis, News Service. Norman, Okla.-based freelance science writer Meg Gordon contributed to this report.

Technology Advances Marking Milestones In Microscopy

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