Today's Image-Analysis Systems Sharpen Focus On Researchers' Needs

David Smith, manager of electronic imaging at Fryer Co. Inc., a Huntley, Ill.-based distributor of optical instrumentation, offers the following suggestions for those considering the purchase of imaging equipment: 1. Define your problem and develop a formal specification of your needs before going out to shop for an image-analysis system. 2. Ask suppliers if their products have the functions your application requires. 3. Determine what the imaging system can do, other than your specific application. 4. Evaluate the system with your samples, in your lab if possible. 5. Find out how many of these systems have been sold and installed. 6. Obtain referrals to scientists who have this system and are doing research similar to yours. 7. Ask what training comes with the system, and the cost for additional training. 8. Before making a purchase, determine how long the supplier has been in business and its ability to provide installation, training, and technical support. --H.G. Today's proliferation of image-analysis and processing equipment has created a bonanza for research scientists. These imaging systems, with their built-in flexibility and customized software, have established themselves as essential tools for acquiring, storing, retrieving, and enhancing data obtained from virtually all experiments conducted through a microscope. An image-analysis system consists of a camera; a processing system comprising a computer; software; and various output and communication devices, including monitors, printers, tapes, modems, and networks. Peripherals like auto-focusing devices, Z-axis stepping systems, filter changers, shutter controls, and computer-controlled staging are also available. The data- collection process can be automated for repetitive operations. "The technology of imaging is relatively young and continues to develop at a rapid pace," says Peter Ramm, president of Imaging Research Inc., a Canadian supplier of image-analysis systems based in St. Catharines, Ontario. "It's only in the last year or two that we have seen the first generation of mature image- analysis software." A number of noteworthy life sciences imaging products are now on the market, he notes. Users have the option of assembling various hardware and software components around their own microscopes, or purchasing a fully integrated imaging system for their research needs. MetaMorph, for example, is a turnkey system from Universal Imaging Corp. of West Chester, Pa. According to Mark Sobiesk, Universal Imaging's sales vice president, the system can provide real-time image processing and enhancement, classify and measure objects, automatically control peripherals, and make time-dependent measurements for studies such as fluorescence recovery after photobleaching (FRAP), the saturation of a fluorescent dye with very intense light. Two other turnkey imaging systems, InCa and InCa++, from Cincinnati-based Intracellular Imaging Inc., also provide quantitative fluorescence microscopy, including single- wavelength and ratiometric analyses. "Innovative hardware engineering has been combined with scientist-designed software to create these very economical and easy-to-use systems," says technical director Jesus Luna. Capturing images with traditional video cameras is giving way to the use of cooled (typically -45xC) charged-couple device (CCD) cameras, which digitize to a higher number of gray levels, allow for variable detector array sizes, and enable images to accumulate for arbitrary lengths of time. Because of these enhancements, cooled CCD cameras significantly increase spatial resolution, temporal resolution, and sensitivity, claims Mark Christenson, a biological applications scientist with Princeton Instruments Inc., located in Trenton, N.J. These features permit the realization of extremely low light techniques, including fluorescence in situ hybridization (FISH)--in which fluorescent DNA probes are used to identify the position of individual genes--and, in the field of single- cell gene expression, the method by which green fluorescent protein (GFP) is produced in a particular cell type under the control of a tissue-specific promoter. In addition to its standard cooled cameras, Princeton Instruments offers a new, high-speed PentaMAX camera, capable of 5 million-pixels-per-second (5 MHz) readout rates, with a simultaneous video signal for focusing or video recording. It is helpful for real-time applications, such as monitoring cytoskeletal dynamics. Also advancing the ability to capture images rapidly is the PXL37, a new, high-speed digital CCD camera system from Photometrics Ltd. in Tucson, Ariz., that can achieve rates of 100 frames per second, according to product manager Jerry Fife. "This product is designed for high-frame-rate quantitative- imaging applications, including calcium ratio imaging, voltage clamp depolarization, cerebral imaging, and neurobiological imaging," he elaborates. Axon Instruments Inc. 1101 Chess Dr. Foster City, Calif. 94404 (415) 571-9400 Fax: (415) 571-9500 Contact: Cheri Harrison Data Translation 100 Locke Dr. Marlboro, Mass. 01752 (800) 525-8528 Fax: (508) 481-8620 Contact: Mike Byerly Fryer Co. Inc. 11177 E. Main St. Huntley, Ill. 60142 (708) 669-2000 Fax: (708) 669-2056 Contact: David L. Smith Imaging Research Inc. Brock University St. Catharines, Ontario Canada L2S 3A1 (905) 688-2040 Fax: (905) 685-5861 Contact: Jill Squires Intracellular Imaging Inc. 1776 Mentor Ave. Cincinnati, Ohio 45212 (800) 401-3553 Fax: (513) 351-7773 Contact: Jesus Luna Media Cybernetics Inc. 8484 Georgia Ave. Silver Spring, Md. 20910 (800) 992-4256 Fax: (301) 495-5964 Contact: Sarita Harris Photometrics Ltd. 3440 E. Britannia Dr. Tucson, Ariz. 85706 (602) 889-9933 Fax: (602) 573-1944 Contact: Michelle Downs Princeton Instruments Inc. 3660 Quakerbridge Rd. Trenton, N.J. 08619 (609) 587-9797 Fax: (609) 587-1970 Contact: Alfred Mottola Scanalytics 40 Linnell Circle Billerica, Mass. 01821 (508) 663-7598 Fax: (508) 663-0150 Contact: David Hitrys Universal Imaging Corp. 502 Brandywine Pkwy. West Chester, Pa. 19380 (610) 344-9410 Fax: (610) 344-9515 Contact: Julie Zolnowski Vital Images Inc. 505 N. Fourth St. Fairfield, Iowa 52556 (515) 472-7726 Fax: (515) 472-1661 Contact: N. Blair Butterfield Software is commercially available to meet many specific requirements. For instance, programs from Axon Instruments Inc., a Foster City, Calif.-based software manufacturer, target the measurement of various analytes and ion concentrations in cells. "Images of concentrations of calcium, hydrogen, and ions can be obtained by acquiring images at each of two wavelengths and calculating a ratio image," explains Michael Delay, manager of scientific business development at Axon. In addition, Axon software allows coordination with electrophysical and ion concentration measurements in order to control cell membrane potential while examining the ion concentration responses over a region of tissue, notes Delay. Neuroscientists, cell biologists, and other researchers say they have found these programs, known as AxoVideo for Macintosh and Axon Imaging Workbench for Windows, to be extremely user- friendly techniques. "We're measuring calcium and sodium ion changes in nerve cells in the striatum and hippocampus," reports Brian MacVicar, a professor of physiology and neuroscience at the University of Calgary, Alberta, Canada. "Not only can we view results online with Axon's software as data are acquired, we obtain a graphic output of the final plot immediately at the end of the experiment. It's a great time saver, as well as an inexpensive solution to our imaging problems." Other software products perform quantitative functions, including counting, measuring, and classifying particles. One example is Image-Pro Plus, a basic imaging package from Media Cybernetics Inc. of Silver Spring, Md., that offers several analytical techniques, such as particle counting, sizing, and brightness measurements, as well as color channel processing. It can be customized with little programming. Similarly, Global Lab Image software from Data Translation in Marlboro, Mass., provides a host of image-analysis features, conducting up to 56 measurements within seconds. Add-on modules that perform specialized functions--such as densitometry, pattern recognition, image compression and conversion, and image databasing--are available, as well, to meet the changing needs of users. Ann Elsner, an associate scientist at the Schepens Eye Research Institute in Boston, uses Global Lab Image to look for changes in the back of the eyes of patients with age-related macular degeneration. "We're doing a characterization study to locate and identify the particles that are related to pathological changes," she describes. To make the study figures meaningful, Elsner must screen 100 patients in each designated category. Without this tool, she notes, her group would be counting away with pencil and paper, and could never effectively examine such large numbers of people. VoxelView/Ultra, VoxelMath, and VoxelAnimatora from Fairfield, Iowa-based Vital Images Inc., constitute a family of cooperative software tools that reconstructs post-acquisition image data into three-dimensional visualizations and animations. "These volume renderings represent 3-D objects as collections of cube-like building blocks called voxels," says Vincent Argiro, Vital Image's president. By comparison, a pixel is a picture element--a tiny, 2-D sample of a digital image. It has a specific location as defined by two coordinates. A voxel, or volume pixel, is a sample that exists in a 3-D grid, positioned by three coordinates, Argiro explains. "We use Vital Image's VoxelView and VoxelMath software to look at the 3-D nature of blood vessels in a hemangioma," says Rick Rogers, director of the biomedical imaging laboratory at the Harvard School of Public Health. "We have determined the 3-D architecture of those capillaries that were growing unchecked." Rogers also used these software siblings to study lipid-filled foam cells in arteriosclerotic plaques. Clearly, biological specimens are three-dimensional structures, and light microscopy is inherently 3-D. At each focal depth, image quality is compromised by light that originates from sources above and below the optimal plane of focus. CELLscan, an integrated image-acquisition system from Scanalytics of Billerica, Mass., contains a novel deconvolution algorithm called Exhaustive Photon Reassignment (EPR) that analyzes the entire set of image planes to reassign blurred or out-of-focus light back to its correct point of origin. This deconvolution process essentially relies on information of how the optical system of the microscope blurs or distorts out-of-focus images. "These images are acquired rapidly under gentle imaging conditions, stored in a computer, and then subjected to the reassignment process at the user's convenience," explains David Hitrys, CELLscan's product manager. This technique yields extremely high contrast and resolution, often revealing structures that have been previously unseen, he points out. Important applications include the dynamic measurement of mitochondrial membrane potentials of living tissue, 3-D localization and quantitation of drug activity within living cells, and radiation effects on human chromosomes. What's on the horizon for image analysis? Imaging Research's Peter Ramm expects to see improvements in the technology for imaging ionic or electrical changes in tissues (optical physiology). There will be new techniques that create images using information other than light intensity--spectral imaging, for example--and quantitative imaging processes will be more widely applied in molecular biology, he adds. Ramm also anticipates advances in macroanatomical imaging: "Now, if you want to show how cells distribute over large areas of tissue, you have to paste micrographs together. Soon, more powerful image analyzers will allow you to assemble many discrete microscope fields of view into large composite images that retain the full resolution of the originals." Adds Mark Underwood, an assistant professor of psychiatry at the New York Psychiatric Institute in New York City: "For nearly a decade, we have been interested in moving past the qualitative description of brain anatomy to focus on the quantification of neuroreceptors using image-analysis techniques." Underwood and his colleagues are studying brain biochemical changes of suicide victims using an Imaging Research MCID system. "The availability of an imaging system on a PC has really allowed for a vast expansion of our capabilities to perform these quantifications." Amid this flurry of image-analysis activity, Ted Inou, Universal Imaging's president, sounds a note of caution: "With numerous imaging manufacturers promoting independent solutions for particular niche markets, the net result could be confusion on the user's part, because no one system can satisfy all applications." However, says Inoue, "Many manufacturers are now working together to develop imaging standards that would allow applications software to work with different hardware systems without the need for custom device drivers for each board. Ultimately, we'll see software vendors concentrating on specific applications, but designing them such that they can be combined to form a complete, coherent system."

Today's Image-Analysis Systems Sharpen Focus On Researchers' Needs

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