Hardware Systems Innovations Bring A 'Renaissance' To Microscopy

Selective microzapping_dubbed laser ablation, a highly accurate technique of micromanipulation that enables researchers to burn off or cut through cellular material without destroying surrounding tissue_offers exciting possibilities as a research tool. However, its benefits have been somewhat compromised because most conventional laser- ablation systems operate at fixed wavelengths, and use open- beam configurations that require great care in maintaining alignment. Often a system must be perfectly aligned by a laser expert, and then isolated from vibration to obtain the desired results. Recently, however, Huntley, Ill.-based Fryer Co. appears to have overcome these bothersome limitations by introducing a tunable ablation laser that features a fiber-optic delivery system. "The position of the microscope relative to the laser is completely irrelevant now," says Robert Nowak, Fryer's laser-systems product manager. "It has absolutely no effect on the unit's operation once the system is put together." In addition, the equipment uses a dye cell that can be tuned within the visible spectrum to more closely match the absorption wavelength of the target structure, achieving a process known as molecularly selective ablation. Called the Bull's-Eye!, Fryer's turnkey system facilitates laser ablation for a wide variety of scientific activities, including developmental biology, cell regeneration, neurology, physiology, genetics, and microsurgery. Burt Evans, a professor of neurobiology and physiology at Northwestern University, is working with hair cells from mammalian inner ears. Using Fryer's tunable laser, Evans is able to ablate certain intercellular structures without damaging these fragile cells or compromising their membrane integrity. "Before Bull's-Eye! came along, there was simply no way to perform intracellular surgery without opening the cell," he says. H.G. Since Antonie van Leeuwenhoek first observed single-celled organisms more than 300 years ago, the microscope has been as ubiquitous a feature of the research laboratory as the test tube for generations of scientists. Perhaps more than any other image, the picture of a technician peering into a microscope has become the accepted symbol for the sciences in general. Although light microscopes have undergone numerous design changes through the years--as is true of many scientific instruments--they have generally retained their characteristic features. In addition, several new hardware systems have expanded the microscope family, bringing a cornucopia of benefits to users. "The current era is sort of a renaissance and revolution in the application of light microscopy to biomedical-research problems," says Fredric Fay, a professor of physiology and director of the bioimaging group at the University of Massachusetts Medical Center in Worcester. "We now have the ability to attain far greater selectivity, such as looking at one specific molecule, or even a particular form of that molecule, within a cell." There also has been a tremendous increase in sensitivity. For example, Fay notes that with the right chemistry, it is possible to measure the activity of a single molecule. And most systems are capable of providing spatial resolutions well beyond what the classical microscopist thought was possible with light microscopy. One advantage of working with light is that imaging can be performed under physiological conditions. "Changes in the distribution or levels of various ions, proteins, or nucleic acids can be recorded as a cell responds to a particular stimulus, allowing some insight to the molecular or ionic changes that are responsible for it," Fay points out. Another advance has made underwater microscopy a practical reality. Melville, N.Y.-based Nikon Inc. has introduced a 60x water-immersion objective that can resolve detail in living organisms at depths as great as 200 microns below the cover glass. Developed in response to the needs of life sciences researchers who previously used oil-immersion lenses to study thick living-tissue specimens, the objective has high transmission and chromatic correction for wavelengths in the near ultraviolet through the red spectrum. Microscope manufacturers are developing a seemingly endless array of new products designed to improve performance as well as be ergonomically pleasing and user-friendly. For example, Olympus America Inc. of Lake Success, N.Y., recently introduced its PROVIS AX70 research microscope, featuring a massive 70-pound, Y-shaped upright stand to optimize stability. Its modular approach offers a comprehensive choice of configurations and varying degrees of automation. A touch-panel microcontroller allows a researcher's fingers to walk through the adjustments, and insertable data cards are available to store all setup information. Olympus touts this feature as especially helpful when instruments have more than one user. Burleigh Instruments Inc. Burleigh Park Fishers, N.Y. 14453 (716) 924-9355 Fax: (716) 924-9072 Contact: Tim Klimasewski Digital Instruments 520 E. Montecito St. Santa Barbara, Calif. 93103 (805) 899-3388 Fax: (805) 899-3392 Contact: Monte Heaton Edge Scientific Instrument Corp. 1630 17th St. Santa Monica, Calif. 90404 (310) 396-9333 Fax: (310) 396-9003 Contact: Linda Wagner Fryer Co. Inc. 11177 E. Main St. Huntley, Ill. 60142 (708) 669-2000 Fax: (708) 669-2056 Contact: Bob Nowak Leica Inc. 111 Deer Lake Rd. Deerfield, Ill. 60015 (800) 248-0123 Fax: (708) 405-0030 Contact: Wayne Buttermore Meridian Instruments Inc. 2310 Science Parkway Okemos, Mich. 48864 (800) 247-8084 Fax: (517) 349-5967 Contact: Carol Genee Meridian Manufacturing Inc. P.O. Box 1204 Kent, Wash. 98035 (206) 854-9914 Fax: (206) 854-9902 Contact: Jan Strelow Molecular Dynamics 880 E. Arques Ave. Sunnyvale, Calif. 94086 (408) 773-1222 Fax: (408) 773-8343 Contact: Steve Nelson Nikon Inc. 1300 Walt Whitman Rd. Melville, N.Y. 11747 (516) 547-8528 Fax: (516) 547-0306 Contact: Al Moline Olympus America Inc. 4 Nevada Dr. Lake Success, N.Y. 11042 (800) 446-5967 Fax: (516) 222-7920 Contact: Reinhard Enders Technical Instruments P.O. Box 2158 Evergreen, Colo. 80439 (303) 674-8780 Contact: Gerald Benham Carl Zeiss Inc. One Zeiss Dr. Thornwood, N.Y. 10594 (914) 681-7752 Fax: (914) 681-7846 Contact: Ernst Keller However, today's upright microscopes often share bench space with their inverted cousins, as scientists realize that sample viewing from the bottom up offers significant advantages. "In addition to accommodating large vessels, such as tissue- culture flasks and other big-specimen containers, an inverted microscope gives clear access to samples for micromanipulation," observes Wayne Buttermore, business- development manager for compound microscopes at Deerfield, Ill.-based Leica Inc. "You can approach your sample from several directions to perform a variety of micromanipulation and microinjection procedures." Leica's DM IRB/E inverted microscope is part of a new generation of Leitz microscopes with infinity optics. Also making its debut is the Axiovert series of inverted microscopes from Carl Zeiss Inc. of Thornwood, N.Y. When coupled to these units, lasers can be used as microsurgical tools for the manipulation of individual cells or cell components. Laser light in the infrared spectral range can interact with a biological material, "trapping" it in place as though it were held by optically created tweezers. Manipulation at the cellular level is rapidly becoming an important research technique, with several intriguing approaches (see accompanying story). To further assist investigators, Burleigh Instruments Inc., based in Fishers, N.Y., has a series of piezoelectric ceramic actuators available for micro-positioning. These units are well-suited for patch clamping, cell penetration, microinjection, electropositioning, and microstimulation, according to Tim Klimasewski, Burleigh's international sales manager. Meridian Manufacturing Inc. of Kent, Wash., increases the versatility of micromanipulation with its orbital-stage systems. These microscope add-ons, currently available for Nikon and Zeiss microscopes, permit the positioning of micromanipulators from any orientation about the optical axis. The orbital stages accommodate up to six individual tool holders. A relatively new kid on the block, little more than five years old, is confocal microscopy. It is a technique that significantly reduces out-of-focus fluorescence light by illuminating just a small volume in the specimen at a time with a scanning laser. Only the light from the illuminated plane reaches the detector, producing exceptionally clear images. A variety of imaging configurations are available from several manufacturers. "We're able to examine mammary glands for cancer cells with our Molecular Dynamics MultiProbe 2001 confocal microscope, following treatment with a precursor and fluorescent probes," says William Brinkley, dean of the graduate school at Baylor College of Medicine in Houston. "The microscope optically sections through our thick specimens, and identifies the location and geometry of the cells of interest. We can obtain really fantastic pictures of areas where tumors might arise." Melvin Schindler, a biochemistry professor at Michigan State University, uses an INSIGHT scanning laser confocal microscope from Okemos, Mich.-based Meridian Instruments Inc. to examine living plant cells. "We have developed techniques to measure tension in the actin network, so that we can detect changes in the cell cytoskeleton due to signaling molecules," he notes. He is planning to extend this technology to study plant-cell response to specific toxic substances in the environment. "We just couldn't make these quantitative measurements in living systems without this instrument," he stresses. "Today's bioscientists are not satisfied with merely acquiring a good qualitative image," notes Gerald Benham, sales and marketing director for Technical Instrument Co. of San Jose, Calif. "They require quantitative data from living specimens at the cellular level. The more sensitive confocal systems become in the future, the more quantitative data will be generated. But we must re-member that confocal microscopy is still in its infancy; we've only just begun." Major advances have been achieved in three-dimensional microscopy. The Edge R400, an upright light microscope from Edge Scientific Instrument Corp., Santa Monica, Calif., provides an elegant technique for obtaining 3-D images of thick specimens, notes Scott Fraser, a biology professor and director of the biological imaging center at the California Institute of Technology. "We can now recognize one object as being distinct from another object in our specimens, and that's a big advantage for us," he emphasizes. Fraser's enthusiasm stems from his ability to see depth in his thick embryonic tissue specimens by taking advantage of the limited contrast available: "Before obtaining the Edge instrument, we had to look at only a very narrow plane, and then computationally superimpose different sections together." According to Edge Scientific executive vice president Christopher Esse, the system utilizes a multiple oblique illumination concept instead of single on-axis illumination, resulting in substantial improvements in resolution, sharpness, contrast, and depth of field. Another instrument that produces 3-D images is the atomic force microscope (AFM). Based on scanning-probe technology, the system measures topography by mechanically moving a sharp probe across the sample to "feel" the contours of the surface. "I believe AFM has five general areas of application," remarks Jan Hoh, an assistant professor of physiology at the Johns Hopkins University School of Medicine. Generating images of surfaces is the main application. "Performing this technique in solution, under physiological conditions, and obtaining resolution on the order of a few nanometers is really exciting," Hoh says. Micromanipulation is another application. For instance, with the probe, an investigator can cut a piece of DNA at a position that is independent of sequence. A third use of the system is measuring micromechanical properties of surfaces, such as cell visco-elasticity. The measurement of intermolecular forces is yet another AFM application. "This capability has tremendous potential in terms of understanding the fundamental aspects of receptor/ligand interactions," Hoh contends, "because you can trigger the interaction of two molecules, an event that is no longer diffusion-limited." In addition, Hoh points out, some AFM users have cleverly developed some beneficial techniques not intended by manufacturers. Researchers have discovered the cantilever probe can be used as a very sensitive thermometer to determine spectroscopic properties, or as a tiny balance to weigh nanograms of protein material. Combining modified inverted optical microscopes with AFM, the BioScope from Digital Instruments of Santa Barbara, Calif., was designed specifically for life scientists. "We have developed scanners that can be immersed in liquids without damage, allowing direct examination of samples in biological fluids," Monte Heaton, Digital's marketing director, points out. "And our TappingMode technique permits easy imaging of delicate samples in air or fluid because the AFM probe lightly taps the samples rather than scrapes their surface." Equally important, according to Heaton, was the development of a highly rigid stage and vacuum mounts for petri dishes, as well as standard microscope slides and cover slips. Without these improvements, he says, the platform simply is not stable enough to attain clear images with nanometer resolution.

Hardware Systems Innovations Bring A 'Renaissance' To Microscopy

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