New Confocal Systems Allow Real-Time Views Of Cells

In just the past few years, technical advances have made possible laser-scanning confocal microscopes able to produce kinetic images of living cells. Depending on the system, neuroscientists and other researchers now can capture images at up to video rates--30 frames per second--or, sometimes, faster. Additionally, they can record those images digitally, or with a video recorder, or with a 35 mm camera. They can view cell events on a monitor or directly through the oculars of the microscope. Leco 3000 Lakeview Ave. St. Joseph, Mich. 49085 (616) 983-5531 Fax: (616) 983-3850 Leica Inc. 111 Deerlake Rd. Deerfield, Ill. 60015 (708) 405-0123 Fax: (708) 405-0147 Meridian Instruments Inc. 2310 Science Pkwy. Okemos, Mich. 48864 (517) 349-7200 Fax: (517) 349-5967 Nikon Inc. 1300 Walt Whitman Rd. Melville, N.Y. 11747-3064 (516) 547-8568 Fax: (516) 547-0306 Noran Instruments Inc. 2551 W. 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Box 6910 Bridgewater, N.J. 08807 (908) 231-1116 Fax: (908) 231-0852 Imaging Technology Inc. 55 Middlesex Turnpike Bedford, Mass. 01730-1421 (617) 275-2700 Fax: (617) 275-9590 The following vendors offer instruments and/or accessories for use in research microscopy and imaging applications. Please call the companies directly for more information concerning their products. Advanced Imaging Concepts Inc. 301 N. Harrison St. Princeton, N.J. 08540 (609) 921-3629 Fax: (908) 274-1974 Alpha Innotech Corp. 14743 Cataline St. San Leandro, Calif. 94577 (800) 795-5556 Fax: (510) 483-3227 Amarel Precision Instruments 78 Schuyler Baldwin Dr. Fairport, N.Y. 14450-9196 (716) 223-2372 Fax: (716) 223-3413 Amray Inc. 160 Middlesex Turnpike Bedford, Mass. 01730 (617) 275-1400 Fax: (617) 275-0740 Biological Detection Systems Inc. 15200 Omega Dr. Suite 105 Rockville, Md. 20850 (301) 990-0100 Fax: (301) 990-8391 Bio-Rad Laboratories Life Science Group 2000 Alfred Nobel Dr. Hercules, Calif. 94547 (510) 741-1000 Fax: (510) 741-1055 Boeckeler Instruments Inc. 3280 E. Hemisphere Loop Building #114 Tucson, Ariz. 85706-5024 (800) 552-2262 Fax: (602) 573-7100 Cell Robotics Inc. 2715 Broadbent Pkwy., N.E. Albuquerque, N.Mex. 87107 (505) 848-0590 Fax: (505) 344-8112 Chui Technical Corp. 252 Indian Head Rd. 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Montecito St. Santa Barbara, Calif. 93103 (805) 899-3380 Fax: (805) 899-3392 Ektron Applied Imaging 23 Crosby Dr. Bedford, Mass. 01730 (617) 275-0475 Fax: (617) 271-1977 Fine Science Tools Inc. 373 G Vintage Park Dr. Foster City, Calif. 94404 (800) 521-2109 Fax: (415) 349-3729 Focus Graphics Inc. 1163 Triton Dr. Foster City, Calif. 94404 (800) 288-6226 Fax: (415) 377-0598 Hamamatsu Photonic Systems Div. of Hamamatsu Corp. 360 Foothill Rd. P.O. Box 6910 Bridgewater, N.J. 08807 (908) 231-1116 Fax: (908) 231-0852 Imaging Technology Inc. 55 Middlesex Turnpike Bedford, Mass. 01730-1421 (617) 275-2700 Fax: (617) 275-9590 Infinity Photo-Optical Co. 706 Mohawk Dr. Suite 15 Boulder, Colo. 80303 (303) 499-6282 Fax: (303) 499-2454 Inovision Corp. 2810 Meridian Pkwy. Suite 148 Durham, N.C. 27713 (919) 361-4609 Fax: (919) 361-5876 When laser-scanning confocal microscopes first began appearing in laboratories in the mid-1980s, many cell biologists found themselves somewhat teased by the powers of the instruments. Capable of extraordinarily sharp images of living cells and their internal structures, these instruments were also too slow in building a digital image, at one or two images per second, to show many dynamic events--potentially, the very reason for looking at living cells. Now, with the faster systems, new kinds of whole-cell investigations have become possible, including, for example, detailed studies of the roles played by calcium and other ions in cell signaling. Calcium-release waves of extremely short duration passing across a cell can now be tracked with these confocal instruments. "We get patterns of calcium liberation which change rapidly, on a time scale of a few tens of milliseconds," says Ian Parker, a professor of psychobiology at the University of California, Irvine. Parker's laboratory is working with oocytes, or immature egg cells, of an African toad, studying second-messenger pathways with a personal computer-controlled system called ODYSSEY from Noran Instruments Inc. of Middleton, Wis. A workstation-based version of the system, called ODYSSEY INTERVISION, is also available. "One of the things we discovered is a phenomenon we've called calcium puffs, which are just brief flashes of calcium, little spots of calcium that suddenly pop up," Parker says. "They last 100 milliseconds or so. With a regular confocal microscope, it would be a matter of chance if you ever saw one of these things at all. And you certainly couldn't follow its course in the way that we can." Besides Parker's Noran system and its more powerful relative, other laser-based confocal instrument systems capable of fast image acquisitions are the ViewScan DVC-250 from Bio-Rad Laboratories of Hercules, Calif.; the INSIGHT PLUS from Meridian Instruments Inc. of Okemos, Mich.; and the RCM 8000 from Nikon Corp. of Melville, N.Y. They range in price from just under $100,000 to more than $200,000, depending on system and accessory choices. The strength of an ordinary confocal microscope, retained by the new, faster systems, is its ability to reject out-of-focus light, or flare, from areas within a cell that are outside the plane of focus. To do this, a point of laser light under computer control is scanned across a specimen, usually fluorescently labeled, at a set depth. The entire plane of focus is illuminated by this beam, but sequentially--point by point--rather than simultaneously, as would be the case with a traditional light microscope. Light returning from the specimen then passes through a pinhole aperture called a spatial filter. It is the spatial filter that defines the confocal microscope; the filter has the effect of screening out light from all but the focal point. The remaining sharply focused light strikes a photomultiplier tube, and, with computer assistance, is used to construct an image, called an optical slice. A large amount of the illuminating light is lost at the pinhole aperture in the confocal process, which is one reason laser light sources have proved so important. Because of the initial intensity of lasers, the in-focus light reaching the photomultiplier tube retains enough brightness to create full images. The confocal microscope can create these images even when the specimen is relatively thick or translucent, and optical slices from different depths can be used to build striking three- dimensional images of a cell. Coupled with the faster image- capture rates now possible, confocal images are opening up many new lines of cell dynamics investigations. "There's a whole world of physiology applications, where people are interested in knowing, `What does this ion do in a living system?'" says Shannon Hall, a Bio-Rad technical specialist. "That ion is most commonly something like calcium or hydrogen. We've known for some time that these types of ions are mediators in physiological interactions, but we really didn't know what was happening. If we can trace them very exactly, then we can have a very good idea what's going on. And that's where confocal comes in." Whether capable of "real-time," video-rate, or even faster imaging, laser-scanning confocal microscopes in this group fall into two categories: point scanners and slit scanners. Each of the two approaches to creating kinetic images has its strengths and weaknesses. For the most part, tradeoffs between resolution and speed are necessary, and the individual researcher's needs will dictate which system is appropriate. The point scanners are, in essence, traditional laser-scanning confocal microscopes with features that allow faster imaging than was previously possible. These include technological improvements in the scanning process as well as computer hardware and software advances in instrument control and data handling. The Nikon and Noran instruments fall into this category. The slit scanners do not use a point of light to scan the specimen, but instead scan with a thin line of light and use a narrow slit opening for detection. This tactic allows the entire field of view to be scanned in a single pass, with correspondingly faster image acquisitions. Additionally, the slit scanners offer the ability to directly view confocal images of the specimen through the eyepiece of the microscope, without the need to display the images on a monitor. The Bio-Rad and Meridian instruments are included in this group. Some loss in image quality does result from using the slit opening as opposed to the pinhole aperture. The Z-axis resolution--the focal depth--is somewhat diminished along the length of the slit. The advantages of slit scanning systems, however, are considerable. The direct-viewing capability makes them easier to use than other confocal systems, more like a familiar light microscope, and the images are in true color--even if that color is fluorescent red and green. Because of their speed, they are often used to quickly screen specimens in large numbers, sometimes for later, more detailed work on a traditional confocal microscope. "It doesn't sound very sexy to say we're looking at more slides than we did with the point scanner," says Brian Matsumoto, director of the integrated microscopy facility of the Neuroscience Research Institute at the University of California, Santa Barbara. "But in terms of experimental analysis, being able to look at a larger sample size may be more critical than the higher resolution," he says. "It may be more significant, because now you can start doing statistical analyses of the cells." Using the Meridian INSIGHT PLUS, institute researchers are performing an array of histopathology studies of cell response to drugs and physical trauma. They sometimes then move to a standard Bio-Rad confocal microscope for follow-up studies, according to Matsumoto. Point scanners, while generally capable of better resolution than slit scanners, must also take the time to make multiple passes across a specimen in order to create an image. To create separate images at video rate--30 frames per second--requires impressive speed, precision, and data handling. Noran's ODYSSEY INTERVISION system and Nikon's RCM 8000 system are both capable of these rates. The Noran system allows the researcher to control the tradeoffs between several critical factors. For example, the image resolution in pixels can be altered against time, so that, at the lowest resolution, 240 images per second are achievable. The Nikon system, developed by Roger Tsien, a professor of pharmacology and chem- istry at the University of California, San Diego, features software designed to counter a type of distortion to which point scanners are subject. "Most systems sweep one way [across a specimen] and then come back to the beginning, or they sweep side to side," explains Ilene Semiatin, a Nikon spokeswoman. "As they go from one side to the other, the mechanics of it are such that the system slows as it gets to the end of a sweep, speeds up in the middle, and then slows again as it gets to the end. Since the images are being created digitally, the fact that there is not a steady speed creates minor distortions." Over the course of the many passes that go into a point-scanned confocal image, the cumulative distortion can be substantial, Semiatin says, and Tsien's software controls the instrument hardware so as to compensate for this effect. Some of these new, faster confocal systems--those with powerful workstations controlling the instruments--are capable of capturing, manipulating, displaying, and storing separate images at video rates. Other systems used professional-quality video systems to capture analog versions of the kinetic cell images. Either way, the separate images are then available for later image analysis. The systems able to capture digital data directly from the microscope retain the most information for later analysis. Video recorder-based systems suffer some information loss, because they must go from a digital source to an analog record and then back again, although researchers say the degradation is not significant for many experimental purposes.

New Confocal Systems Allow Real-Time Views Of Cells

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