by Jeffrey M. Perkel
Imagine you want to understand the rules of American football. If a friend showed you three snapshots taken during one game, would you be able to suss out what's going on? Probably not. But, if you were able to watch the game live, things likely would be a whole lot clearer. That, in a nutshell, is the rationale behind live-cell imaging, a fast-growing segment of the microscopy
Marketplace, says Claire Brown, director of the imaging facility at McGill University in Montreal, Quebec.
"A picture is worth a thousand words, so what's a movie worth?" Brown says.
The key concept, explains Stan Schwartz, vice president for global training and education at Nikon Instruments, is dynamics. Dead men tell no tales, and neither do dead cells. "People have looked at stuff that's dead and sliced and stained—everyone has looked at everything there is to look at that way already," he says. "Today's question is about the process, the molecular process of living cells. To [address] that, they have to be alive."
Of course, live-cell imaging isn't really new, per se; for all intents and purposes, it's really just time-lapse photography. Schwartz calls it six-dimensional microscopy: the three spatial dimensions plus wavelength, time, and position. Any number of microscopy approaches may be used, from brightfield to confocal to TIRF. Yet in all cases the images are snapped periodically, from several times per second to every quarter-hour or so, for anywhere from seconds to minutes to days.
Brown, who authored a practical overview of live cell microscopy techniques for the Journal of Cell Science in 2009 [1], estimates that live-cell imaging accounts for about half of her facility's work – up from 5-10% in 2005. The technique, she says, is really no more challenging than standard fixed-cell microscopy—if you have the right tools. "I wouldn't say it's difficult, but I would say you need to be careful and do your [homework]."
You'll also need a different set of equipment.
That's because live cells are, well, alive—and for an experiment to be scientifically useful, they have to be kept that way. Standard microscopy equipment wasn't really designed with that goal in mind. Dead cells, for instance, don't care how much light hits them (though their fluorophores might); but live cells do, and they may react accordingly.
"It's like throwing a spotlight into a cave to observe bats," says Schwartz, "You may not necessarily be observing how they behave in the dark."
In its September 2009 "MicroImaging Newsletter," Carl Zeiss MicroImaging lays out "Five Basic Requirements" for live-cell imaging work. Researchers, the newsletter says, need (1) a method for specifically labeling the protein or cell structures of interest; (2) a way to illuminate those cells with a minimum of phototoxicity; (3) a highly sensitive detection system capable of capturing the relatively few photons such low-intensity illumination yields; (4) an incubation system that can keep the cells alive and physiologically "happy" during the experiment; and (5) a way to keep the cells in focus over time.
Labeling
Many fluorescent live-cell imaging experiments use genetically encoded fluorophores (i.e. green fluorescent protein—GFP, and its kin), because they require no input chemicals (such as fixatives) to work and can be tagged to specific proteins or cell structures of interest. However, membrane-permeable live-cell reagents are also available, such as Life Technologies' MitoTracker and Fura-2 AM (a calcium indicator). Enzo Life Sciences offers some 40 kits to probe cellular physiology under its CELLestial™ trade name, according to Chief Scientific Officer, Wayne Patton. Among the newest is the Cyto-ID™ Autophagy Detection Kit, which lights up cells that are essentially digesting themselves from the inside out in response to such environmental perturbations as nutrient deprivation, pathogen attack, or toxic drug treatment.
Illumination
Illumination is a critical factor in live-cell imaging, especially if that imaging is to proceed for longer than a few minutes. Most mammalian cells are never exposed to light, Brown notes.
According to Michael Davidson of the National High Magnetic Field Laboratory at Florida State University (Davidson also runs the Molecular Expressions website that contains, among other things, many useful resources for live-cell imaging), standard widefield microscopy systems use arc lamps as opposed to lasers, and some fraction of those lamps' output, especially in the ultraviolet and infrared regions, can be toxic. For live-cell work Davidson recommends a gentler approach.
One option is to use a laser. Most laser-based systems use fixed wavelength lasers that can excite narrow sets of fluorescent dyes. Leica Microsystems' TCS SP5 X confocal microscope, however, couples a tunable white-light laser (470-670 nm), an acousto-optical beam splitter (AOBS), and sensitive spectral detectors, so users can excite the fluorescent markers in their samples at precisely the wavelength desired and efficiently detect the fluorescence signal at its optimum emission range, whatever the dye being used.
Alteratively, consider light-emitting diodes (LEDs). "You can use any light source for fixed cells, but it makes sense to use a gentler light source like LEDs for live cells," says Megan MacNeil, a product marketing manager at Zeiss.
Researchers can also use the relatively gentle fluorescent illumination of spinning-disk confocal imaging. Unlike traditional laser-scanning confocal microscopy, in which a high-intensity laser is rasterized across the sample, spinning disk systems dilute that light by passing it through a rapidly spinning disk peppered with tiny pinholes, thereby illuminating many points on the specimen simultaneously. Not only is such a strategy less phototoxic, it is also faster than laser-scanning methods.
Spinning disk technology, Davidson notes, isn't new, but it has evolved. In particular, he says, many live-cell systems incorporate the new Yokogawa CSU-X1 spinning disk system. Incorporating two synchronized disks—one containing pinholes, the other containing microlenses—the system, Davidson explains, "illuminate[s] the sample at low intensity. You can bathe the cells in this stuff longer without killing them and take images much, much faster [than with laser-scanning confocal imaging]." In fact, the system theoretically can capture 5,000 frames-per-second (though in practice, such speed is neither practical nor needed, Davidson says). Zeiss' turnkey live-cell imaging Cell Observer® SD system incorporates the Yokogawa system, for instance.
Detection
To capture the fluorescence arising from such relatively weak excitation, you'll need a sensitive detector. One of the most popular for live-cell imaging is an electron-multiplying CCD (EMCCD) camera, such as those from Andor Technology and Photometrics. According to MacNeil, the quantum efficiency of EMCCDs can exceed 90% (that is, 90% of photons that hit the CCD will be detected), compared to 65% for standard CCDs. Alternatively, some systems (including Zeiss' LSM 780) use gallium-arsenide-phosphide photomultiplier tubes, whose quantum efficiency is around 45% (compared to 25% for standard PMTs).
Environmental Control
To keep the cells alive while they are imaged, you'll need some sort of environmental control system. These can range from small stage-top perfusion chambers to large plastic boxes that essentially encompass the microscopy setup. Though the former is less bulky, the advantage of the latter approach is that all the components are heated to the same temperature, reducing thermal "drift" (focus problems that arise when different components in the microscope are at different temperatures).
Olympus' VivaView system is a turnkey live-cell imaging system that follows the microscope-in-a-box approach. According to Product Marketing Director Edward Lachica, the VivaView is an inverted microscope embedded in an incubation system. Users can control the incubator's temperature, gas mixture, or humidity. But according to Lachica, what makes the VivaView unique is its eight-position plate carousel.
Like most live-cell systems, the VivaView enables users to select specific positions to image over and over. At each position, they can take a brightfield image, as well as multiple fluorescent images at different z-positions. And, they can cycle from position to position and plate to plate for as long as the experiment runs; according to Lachica, experiments can last six days or even longer. "We have a unique opportunity to capture a lot of different scenarios in a very, very stable environment," he says.
Autofocus
The final component of a successful live-cell imaging experiment is an active autofocus system. As cells move and grow, they can move out of focus; active autofocus systems repeatedly query the underside of the coverslip and adjust optical elements to keep the distance from the objective to the sample constant. All the major microscopy vendors include such devices, such as Nikon's Perfect Focus System and Leica's new Adaptive Focus Control system (part of the company's DMI6000B, which anchors that company's live-cell portfolio for widefield and confocal systems).
Buying Advice
If you're going to make the move to live-cell imaging, be prepared to spend some extra money; environmental control systems, software, detection hardware, and optics all cost money. Both off-the-shelf and customized systems are available, of course—a few were mentioned in this article—and a wide range of third-party vendors exists to help.
But don't forget the basics. For instance, to image live cells in media, you're going to need dedicated objectives. A number of companies sell the water-immersion objectives required to make good optical contact with your aqueous specimens. Nikon, for instance, offers a 60x water-immersion lens with 1.27 NA and a 170-um working distance. "That is a huge, huge improvement over [standard] 1.2 NA objectives in resolution and brightness," Schwartz says.
But water can evaporate pretty quickly at 37°C, causing optical aberrations. Apart from using immersion oil, one option is to use glycerol immersion lenses; glycerol's refractive index still isn't quite as good a match as water to the cellular milieu, but it doesn't evaporate quite so fast, either. Alternatively, Leica offers a Water Immersion Micro-Dispenser that keeps the immersion fluid levels topped off. Says Sebastian Tille, head of Leica Microsystems' Widefield Imaging business, "if your water evaporates, that's the end of your experiment."
Reference
[1] M.M. Frigault et al., "Live-cell microscopy—tips and tools," J Cell Sci, 122:753-67, 2009. [doi:10.1242/jcs.033837]