Advancements Enable Scientists To Use Their Microscopes In New Ways

Ways Author: Holly Ahern In many areas of neurobiology and cell biology, researchers who a decade ago were confined to studying stained tissues are today using their microscopes in new ways to directly observe dynamic events as they occur in living cells. Although the optical systems of microscopes have not changed dramatically over the last quarter of a century, new methods of acquiring images and processing microscopic infor-mation have indelibly changed the way that scientists view the microscopic world. Particularly in signal transduction re-search-the area dealing with how cells send and receive chemical messages-the development of sensitive and specific fluorescent dyes combined with light-sensitive TV cameras and affordable computer imaging systems have led to an explosion of interest on the part of cell biologists and neurobiologists alike. "In 1985, a [Medline] database search of the scientific literature for references related to intracellular calcium yielded less than 10 papers," says Eric Gruenstein, a professor of molecular genetics and biochemistry at the University of Cincinnati Medical School and president of Cincinnati-based Intracellular Imaging Inc. "By 1990, that number had increased dramatically, and is now in the range of hundreds of papers per year." INTEGRATED IMAGES: The CELLscan system from Scanalytics/CSPI produces high-resolution, 3-D representations of specimens. "Essentially, technological advances in physics and chemistry have provided us with opportunities to directly observe living cells and their intracellular activities," notes William Mantulin, director of the Laboratory for Fluorescence Dynamics, a National Institutes of Health research technology center based at the University of Illinois in Urbana-Champaign. Since Roger Tsien, a Howard Hughes Medical Institute investigator and a professor of pharmacology and chemistry at the University of California, San Diego, developed the first of the calcium-detecting fluorescent dyes (BAPTA, followed by fura-2, indo-1, and others) in 1975 and into the 1980s (R.Y. Tsien, Annual Review of Biophysics and Bioengineering, 12:91-116, 1983), researchers have achieved a window on the cell. Using fura-2 with a fluorescence-microscopy system, such as the Attafluor Digital Fluorescence Analyzer from Carl Zeiss Inc. Microscope Division of Thornwood, N.Y., scientists can directly observe oscillations in intracellular calcium levels. These may appear as spirals or spikes of the ion as cells respond to stimuli such as hormones or drugs. Neurobiologists, for example, can observe complex calcium signaling events, such as synchronized calcium spikes and waves, in neurons as the neurons are stimulated. "Now that we've observed these intracellular changes, we need to find out why they occur and if these fluctuations in ion concentration are physiologically important," reports Gruenstein. Because of the research efforts of physicists Ernst Abbe, Carl Zeiss, and others in the latter part of the 19th century, microscopes achieved their theoretical resolution limit (where two microscopic objects are clearly discernible) by the turn of the 20th century. Since that time, the image-forming optical systems of compound light microscopes have remained virtually the same. Light waves that travel through a specimen are collected by lenses to create a magnified virtual image of the microscopic object. Simple laws of physics limit how much an image can be magnified and still be clearly seen: Unlimited magnification is not possible without an associated loss of resolution. Major manufacturers-including Olympus America Inc. Precision Instrument Division of Melville, N.Y.; Nikon Inc. Instrument Group, also based in Melville; and Carl Zeiss Inc.'s Microscope Division-make microscopes for every conceivable application. In addition to the standard compound light microscopes, optical systems for phase-contrast (PC) and differential interference contrast (DIC) microscopy alter the ways in which light waves are collected by the lenses, creating higher-contrast images rather than greater resolution. Fluorescence microscopes detect the low-energy wavelengths emitted from fluorescent objects or dyes, which again improves contrast while the resolving power remains the same. Many microscopes can be equipped for more than one type of optics. Nikon's Opitphot-2 multifunctional research microscope can be user-configured for simultaneous fluorescence and DIC imaging. Electron microscopes achieve high-resolution images that can be magnified as much as a million times, but at the expense of the specimen, which is first subjected to a harsh and lethal fixation and staining process. In all of these methods, the virtual image is subject to one final set of optics for the detection process. Until the 1980s, the detection system most often associated with microscopes was the human eye. "Eyes are very good spectrometers [detecting differences in color], but they are not very good photometers in the sense of quantitatively distinguishing shades of gray," explains Gruenstein. In non-color images, such as what one sees when observing a living cell with a conventional light microscope, it is often impossible for the viewer to detect subtle and even not-so-subtle shade differences, created as light passes through various parts of cells. One solution is to chemically fix the cells and stain them with colorful dyes to generate contrast for the eye to perceive. Fixed and stained cells, however, are dead, and thus they are not observed in their natural state. An alternative solution is to use a better detection system. "Detection systems are sophisticated imaging devices with arrays of sensors that can create high-resolution, three-dimensional images of microscopic objects," says Stephen DeGraw, marketing manager at Oncor Imaging in Gaithersburg, Md. "They are not just cameras anymore." Low-light-level charged-couple device (CCD) cameras such as the PentaMAX digital camera from Princeton Instruments Inc. of Trenton, N.J., discern light at much lower levels-such as the weak light emitted from a fluorescently stained sample-than the human eye. Even with a sensitive video imaging system, quantitative analysis of minute detail is best done if the image is considered numerically. "Bringing out spatial variations in images that might be hidden or difficult to detect is best accomplished by treating the image mathematically," states Gruenstein. Digitizing the image into numerical data points is achieved with a computer, usually interfaced with a video camera via a digitizing frame grabber. Once digitized, stored images can be processed using software that allows researchers to observe the activities of living cells. Where once applications-such as watching nerve cells growing, locating and counting subcellular organelles, or observing the ebb and flow of intracellular ions as cells transduce messages-required the number-crunching capabilities of a mainframe or mini-computers, powerful PCs such as IBM's Pentium PC and the Apple Macintosh's PowerMac bring imaging applications to the benchtop. "In the past, cameras and other detection devices have had very good quantitative capabilities with high resolution, but because of the vast amount of information to be processed, detection systems have been slow," notes DeGraw. "Improvements in the electronics and computing power allow for faster readout of the information and better imaging capabilities." Creating and manipulating digitized images requires software that serves as the interface between the image and the investigator. For image analysis and measurement applications, Jandel Scientific Software of San Rafael, Calif., offers two PC-based software packages, SigmaScan 2.0 and SigmaScan Pro 2.0. Both packages are compatible with the new Microsoft Windows 95 interface. Images can be scanned in the program and loaded as files of digitized data, or, with SigmaScan Pro, live video images can be captured from a screen with a video camera and "frame grabber." IMAGE INTERFACE: Jandel Scientific offers SigmaScan, middle screen, and SigmaScan Pro software for PC-based image analysis and measurement applications. In SigmaScan, users can collect measurement data from any digitized image. The package offers users 44 different measurements for parameters such as angles, shape, and intensity, as well as manipulation and enhancement features. SigmaScan Pro is an automated image-analysis package that analyzes color and gray-scale images captured from live video, input from scanners, or loaded from a disk. With this package, researchers can measure and count objects in the image, find image intensities, and quantify morphometrics (measure size and shape). Axon Instruments Inc. in Foster City, Calif., also sells imaging software for PC and Macintosh computers. Axon Imaging Workbench, for example, is an image-analysis package specific for intracellular imaging of ions, such as calcium and cyclic AMP for cell-signaling studies. As a research tool, microscopy has given way to imaging, a combination of microscopy techniques, chemistry, physics, and computer science. Imaging requires imaging systems, composed generally of a microscope, a detection system (such as a cooled CCD camera), and software to permit processing of the digitized image. For studying nerve cells with fluorescent probes, for example, imaging offers advantages over conventional microscopy. "The low-light-level cameras commonly used to study living cells allow you to detect very weak signals," notes neurobiologist Greg Lnenicka, an associate professor of biology at the State University of New York, Albany. "Once the image is digitized and in the computer, the data can be processed to quantify various aspects of neuronal structure and process. For instance, one can follow changes in intracellular ion concentration, membrane potential, or the structure of fine neuronal extensions." One problem inherent in microscopy systems is depth of field. Because many specimens are more than one cell layer thick, lenses may collect light from more than one focal plane, creating an out-of-focus "haze" of light that obscures the desired image. Solving this sort of problem is custom-made for a computer, which can manipulate the numerical data to bring that light back into focus, or remove the out-of-focus light from the equation altogether. The CELLscan system from Scanalytics/CSPI of Billerica, Mass., is an integrated image-analysis system based on an algorithm developed by Fredric Fay at the University of Massachusetts Medical Center at Worcester, Mass., and independently by David Agard and John Sedat at the University of California, San Francisco (for a review, see R.Y. Tsien, Chemical and Engineering News, July 18, 1994, pages 34-44). With this system, images are obtained with a fluorescence microscope at sequential focal depths until a through-focus series of images is achieved. These images from each focal plane are sent to an array-processing computer, which reconstructs the signals into an image. The result, according to Scanalytics, is a high-resolution, three-dimensional representation of the specimen. Confocal imaging systems link confocal microscopy, a technique dating from the 1950s, to computers as an approach to eliminating out-of-focus light from thick or heavily stained specimens. In this approach, the image is illuminated one point at a time with a laser or other source of fine, cohesive light. Image-forming light rays are collected through a pinhole-sized aperture, which physically rejects the out-of-focus light from other areas of the specimen. Confocal microscopes can be widely applied to studies in the life sciences, including intracellular ion imaging and cell-structure analysis. "Confocal microscopy allows you to optically section a tissue," explains Lnenicka. "Then the computer recombines all of the optical sections into a final three-dimensional image. Optical sectioning can be achieved with a conventional microscope; however, the real advantage of the confocal microscope is elimination of the out-of-focus flare." The LSM 410 inverted laser-scan microscope from Carl Zeiss Inc. is a confocal imaging system that runs on a Windows interface. With this system, the scanning format and the time series can be set according to the user's preferences, with special features to capture very rapid changes in cells, such as those that occur in cardiac muscle cells or other active cells. The CLSM 2010 system from Molecular Dynamics Inc. of Sunnyvale, Calif., is an inverted confocal laser-scanning microscope system that applies patented confocal sectioning and three-dimensional reconstruction techniques for high resolution and sensitive imaging. Bio-Rad Laboratories of Hercules, Calif., offers the MRC-600 and MRC-1024 confocal laser-scanning imaging systems, with improved photomultiplier tube detectors for enhanced sensitivity. The growth of signal transduction as an area of research has led to several developments in fluorescence microscopy for intracellular ion imaging. Several companies now market both Windows- and Mac-based imaging systems specific for making quantitative measurements of fluorescence in cells, the apparent hallmark of cell-signaling events. Universal Imaging Corp. of West Chester, Pa., carries two PC-based image-analysis packages. The MetaMorph system is a general-purpose device designed for a wide range of microscope applications, including measurements, time-lapse imaging, three-dimensional reconstruction, image enhancement and sharpening, and real-time background subtraction. MetaMorph includes a ratio imaging option of intracellular imaging, as well. Universal Imaging's MetaFluor imaging system is specifically designed for quantitative ion imaging and ratio determinations, for quantitative fluorescent measurements in cells. Oncor Imaging's Ratio ++ is a Mac-based interactive interface specifically designed to support intracellular imaging applications. Inovision Corp. of Durham, N.C., offers the video-based IC-500 imaging system and software products, such as the Ratiotool package for high-resolution imaging. Inovision's systems are capable of performing several image manipulations, such as real-time fluorescence ratio imaging, automated rare-event screening, morphometry, and densitometry. QUANTITATIVE MICROSCOPY: The InCa++ system from Intracellular Imaging for measurement of intracellular fluorescence, especially analysis of cytoplasmic calcium. The InCa++ and InCa systems from Intracellular Imaging are integrated calcium imaging systems for quantitative fluorescence microscopy of ratiometric (InCa++) and nonratiometric (InCa) fluorescent dyes inside of cells. Particularly for calcium imaging, "image analysis and image enhancement make quantitative results possible," comments Gruenstein. Researchers agree that being able to manipulate images simplifies their research and, in some cases, makes it possible. "Having a digitized image makes analyzing microscopic data much easier," says Lnenicka. In some cases, image enhancement enables a scientist to see things that might not have been detected without the aid of the computer. "Enhancing images to improve contrast has made it possible for cell biologists to observe intracellular structures [such as microtubules] inside of living cells. This wouldn't be possible without image-analysis techniques." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. (The Scientist, Vol:9, #21, pg.18-19 , October 30, 1995) (Copyright © The Scientist, Inc.)

Advancements Enable Scientists To Use Their Microscopes In New Ways

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