Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Suppose researchers want to understand how a particular protein goes from point A to point B in a cell. They could simply kill and fix cells at specific time points in the process, image the cells microscopically, and try to work out what happened. Or, they could keep the cells alive and observe the protein as it goes about its business.
The latter approach, called live cell imaging, is certainly conceptually simpler — it’s a whole lot easier to reconstruct behavior from video footage than from disconnected snapshots, after all. Yet it also is more technically demanding, says Claire Brown, assistant professor of physiology at McGill University and director of the Life Sciences Complex Imaging Facility.
Fixed, dead cells, don’t particularly care how they’re treated, she notes. But live cells most certainly do, responding (often negatively) to environmental fluctuations in such variables as temperature, media composition, pH, and light.
One colleague, Brown recalls, was imaging mouse embryos when the temperature dropped unexpectedly one degree, to 36˚C. “The heart stopped beating,” she says. “Once they got the temperature back up to 37˚C, the heart started beating again.” Another researcher, who was trying to image C. elegans under a stereomicroscope, discovered the worms are not big fans of the microscopy spotlight. “They swam away because the light was presumably too bright,” she says. “We had to tone down the light exposure to make them stay still.”
Microscopy vendors now offer a broad range of tools to facilitate live cell imaging, from dedicated microscope systems and optical components to add-on modules that can hold environmental variables constant. Indeed, it’s never been easier to record the secret lives of cells. If you’ve been waiting for the right time to incorporate live cell microscopy into your research, that time is now.
Live cell applications
Brown says live cell microscopy accounts for about half the imaging work done in her facility at McGill—a fraction she wishes were higher. “There’s just so much more information you can get by doing things live,” she says—studies of cellular protein trafficking, cytoskeletal dynamics, and signaling, for instance.
Brown’s own research involves the dynamics of cellular motion, “how cells move,” as she puts it. For instance, her lab studies the assembly, regulation and dynamics of focal adhesions, the cellular “feet” that attach cells to their local substrate and help determine whether cancer cells will invade surrounding tissues. She applies a range of microscopy platforms to that problem, from widefield fluorescent microscopes to laser-scanning confocals to total internal reflection fluorescence (TIRF) microscopes. “TIRF is very good, because you’re only exciting the bottom 100 nm of the sample,” she says.
Brown says most of the significant advances in live cell imaging that have caught her attention over the past few years have occurred on the reagent front, where clever researchers have developed probes that can reveal not just a protein’s location but its function and physics. Researchers have, for instance, developed fluorescent biosensors responsive to GTPase activation and phosphoinositide concentration.
One team, led by Martin Schwartz at the University of Virginia, developed a fluorescence resonance energy transfer (FRET)-based biosensor for protein tension that harnesses the properties of a spider silk protein as a kind of spring. When the protein is relaxed, fluorescent proteins on either side of that spring are close enough to interact and produce a FRET signal. But when the protein is under tension, FRET across the spring drops off, indicating the two domains are pulling apart [1]. Schwartz’s team used this system to study the forces impacting vinculin, a cytoskeletal protein involved in focal adhesions.
At the National High Magnetic Field Laboratory at Florida State University, nearly three-quarters of microscopy experiments are live cell, according to lab director Michael Davidson. The lab studies fluorescent protein dynamics using some 25 live cell systems, Davidson says, from spinning-disk-based confocal microscopes to super-resolution systems; the choice of which to use is largely application-based.
Live cell experiments can last anywhere from microseconds to days, Davidson explains. “It depends on the speed of the dynamics of what you’re looking at.” Calcium signaling, for instance, occurs incredibly quickly. “You need to be able to get 10 to 100 shots per second with those guys,” he says. “If you’re looking at something like focal adhesions, you can take a shot every five minutes and not see much change.”
Just about any good microscopy camera will suffice for such slow imaging applications. But Davidson credits new detection systems, especially CMOS cameras from the likes of Andor and Hamamatsu Photonics, with enabling ultrafast imaging. Those cameras can capture “thousands of frames per second,” but are not yet widely used, he says, noting they have “a lot of potential, but they are still in their infancy.”
New microscopy systems
For imaging over the long term, cells cannot simply be left exposed to ambient air; they must be kept in special chambers—basically small incubators—that allow control of temperature, humidity, and carbon dioxide levels. Davidson’s lab uses one such system from Bioptechs, and a variety of alternatives exist.
EMD Millipore’s CellASIC™ ONIX system is a microfluidic platform intended for use on inverted microscopes that enables researchers to automatically control reagent addition and atmospheric conditions in each well of a dedicated microfluidic plate consumable. (Several configurations are available, including designs for bacterial, yeast, and mammalian cells.)
One application, says Philip Lee, research scientist fellow at EMD Millipore, is monitoring cell response to different drugs. Normally, that requires removing the plates from the incubator, adding the compounds, and putting them back in the incubator. But cells don’t necessarily respond well to such environmental changes.
“The most useful thing about the ONIX is it automates the process,” Lee says. “You can put your cells and consumables on the microscope, set up the experiment, let it run and capture the data. That’s what our customers are doing that couldn’t be done with alternate approaches.”
Another key requirement of long-term, live cell imaging is focus control. Suppose you are imaging a particular cell, or cell region, over several hours. If the system loses focus due to “focus drift” or thermal instability, the dataset might be ruined. Autofocusing systems like Nikon’s Perfect Focus and Leica’s Adaptive Focus Control correct for these problems.
Nikon released its third-generation Perfect Focus system at the 2012 Society for Neuroscience meeting. Perfect Focus maintains focus by more or less simultaneously imaging a discrete autofocus path in addition to the sample’s optical path, ensuring that the distance between the two remains constant. “We monitor focus every five milliseconds,” says Stephen Ross, the company’s general manager for the Product and Marketing Department.
Ross says the third-gen Perfect Focus “more than tripled the [system’s] offset distance”—that is, the distance between the reference and focal planes—thereby enabling deeper imaging applications into, say, tissues. Nikon also released a second Perfect Focus system for multiphoton microscopes, thereby bringing the autofocus technology to in situ brain research, as well.
Leica’s Adaptive Focus Control system is available on the company’s TIRF microscopy systems, says Sebastian Tille, director of widefield imaging at Leica Microsystems. “Even if you work over several hours, you can be sure you’re still focusing on the same part of the sample,” he says.
Also of importance in live cell imaging is phototoxicity. Most cells in the body are never exposed to light, says Brown, and the more light directed at a cell, the less happy it will be. Spinning disk confocal systems (such as Olympus’ CellVoyager™ CV1000 and the Zeiss Cell Observer SD) are a popular choice for live cell imaging, as they effectively dilute imaging light through thousands of pinholes. A newer option is “light sheet microscopy” (commercialized in such systems as Zeiss’ Lightsheet Z.1), which uses multiple orthogonal objectives to illuminate thick samples (such as a zebrafish embryo) with a planar “sheet” of light, thereby reducing phototoxicity and increasing imaging time relative to confocal approaches.
Leica and Nikon now offer ultrafast tandem scanning heads for their confocal microscope lines to bring reduced phototoxicity to laser-scanning systems. Nikon’s A1R confocal microscopes and Leica’s new TCS SP8 microscopes are both available with dual galvo-resonance scanning heads, which can be operated independently of one another.
According to Ross, the key feature of tandem scan heads is that, whereas the galvo head scans relatively slowly, the resonance head scans exceptionally rapidly. Thus, says Ross, researchers could, say, photoactivate a set of fluorescent proteins in the endoplasmic reticulum of a cell with the galvo scanner and then image their trafficking dynamics at more than 30 frames per second full-frame (or 400 fps in “band” imaging mode) with the resonance scanner.
Leica launched its TCS SP8 platform in 2012, says Tille, where it joined the company’s DMI6000B line of live cell systems. Besides the optional tandem scanning head, which can scan at up to 428 fps, the TCS SP8 also includes a new hybrid photodetector called HyD™ and a tunable white-light laser with acousto-optical beam splitter (AOBS).
“With SP8 you have the ultimate freedom of selecting excitation and emission wavelengths and can tune and tweak that so it optimally covers your experiment,” Tille says.
Live cell super-resolution microscopy
One growing application in the live cell arena is its union with super-resolution microscopy. Previously, most super-resolution techniques—especially those, like photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), that rely on multiple rounds of turning specific fluorophores on and off to localize each point—were too slow and phototoxic for use with live cells. Recently, though, researchers have begun figuring out ways to circumvent those issues.
Davidson’s lab routinely performs live cell PALM imaging, he says, but mostly of relatively slow cellular processes, such as cytoskeletal imaging, which don’t conflict with the slow scan rate of the imaging method. “It’s technically challenging, but not all that challenging,” Davidson says.
Leica’s SR GSD system, introduced in 2011, supports “ground-state depletion microscopy followed by individual molecule return” (GSDIM) super-resolution imaging. Unlike PALM and STORM, which use laser energy to turn on photoswitchable markers, GSDIM actually does the opposite: It uses its laser to make the fluors dark and then allows them to reactivate automatically and stochastically. “That’s more direct,” Tille says, “and a very solid method.”
Tille says one of the strengths of the Leica SR GSD is stability. With localization methods like GSDIM—or PALM and STORM for that matter—many thousands of frames must be captured and integrated to produce the final image. The crucial point, he says, “is you need to ensure that during acquisition, there is no drift.” The SR GSD system achieves that goal using its SuMo (“suppressed motion”) stage to keep the sample and objective “tightly linked,” Tille says.
STORM, available through Nikon, is also amenable to live cell imaging. In 2011, for instance, Xiaowei Zhuang, who invented the technique, described one way to use it to collect 3D datasets of transferrin endocytosis in live cells [2].
We want to elucidate the dynamic events that go on inside cells. What are proteins doing and what are their interactions? With live cell imaging, researchers can finally begin to answer those questions.
References
[1] C. Grashoff et al., “Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics,” Nature, 466:263–6, 2010.
[2] S.A. Jones et al., “Fast, three-dimensional super-resolution imaging of live cells,” Nat Meth, 8:499–505, 2011.
The image at the top of the page is from Zeiss.