Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Live-cell imaging isn’t just about taking pictures of live cells (as the name would imply)—the term has come to mean imaging of cells over time, whether that’s a thousand frames per second for a second, or once every 10 minutes for a week. For many in cellular and molecular biology, live-cell imaging has become a valued tool in the toolbox, if not the mainstay of their research.
If you want to do live-cell imaging yourself, you’ll need more than just a microscope. Additional enabling equipment typically is needed to keep the cells happy and ensure you capture the images you want. Here we look at some of your options.
Why live imaging?
Much can be learned by taking cells out of an incubator and photographing them under a microscope every few minutes or hours, and then returning them to their moist, warm home. Observing slow processes such as cell division, for example, may be well served by such a procedure. Yet it’s not always easy to find the same cell in the same field of view, and the very process of moving the culture can disrupt delicate processes, as well. For faster processes, such as cytoskeletal remodeling, this approach isn’t even an option.
If a picture is worth a thousand words, a movie is worth a million—so says David Spector, director of research at Cold Spring Harbor Laboratory and co-editor of “Live Cell Imaging: A Laboratory Manual.” He explains: “There are so many things you can learn from watching the actual dynamic aspects of what you’re studying over time. That continuity provides you a lot of insight that you just don’t get from looking at [a] fixed time point.” Imagine two proteins that are separate at one time and together at a later time—“what’s the choreography of how they move and find each other in three-dimensional space?”
To make movies, researchers typically put a glass-bottom plate or dish containing the culture onto the stage of an inverted microscope and leave it there for the duration. If the scope is equipped with a computer-controlled motorized stage and automatic focusing and tracking capabilities, multiple areas of interest can be located just once and then revisited at will.
Happy cells in boxes
The length and type of experiment helps to dictate the level of environmental control needed to keep the cells happy. Krzysztof Hyrc, manager of two imaging cores and assistant professor of neurology at Washington University in St. Louis, performs calcium imaging. He notes, “if all you want to do is a quick-and-dirty experiment, you do it at room temperature without caring about the atmosphere.” Just make sure the medium is bicarbonate-free, otherwise the pH will go up very, very quickly without a controlled CO2 environment.
The next step would be to keep the cells at 37°C, says Hyrc. For that, there are several options. Perhaps the simplest is to fashion a DIY solution, such as “a simple heating coil with a feedback loop,” Hyrc suggests. “Cover it with silicone so that it doesn’t interfere with the media, submerge it in the dish and you’re ready to go,” he says. “I’ve been doing this sort of experiment on a temperature-sensitive mutant that changes its properties between 34° and 37°. The experiments can certainly last for an hour.”
There also are commercially available stage-top holders that can maintain plates and dishes at a constant temperature. Some of these—from Tokai Hit, for example—are available as a closed chamber with a gas mixer. Or researchers can instead purchase a less expensive chamber that uses pre-mixed gas (such as 5% CO2). But either way, warns Hyrc, “make sure the gas is humidified and warmed up—this way you can operate for a long time.”
Humidity is important because “quite an amazing amount of evaporation” can occur at 37°C, says Kate Luby-Phelps, resource director of the University of Texas Southwestern Medical Center’s live-imaging facility. “And this increased tonicity [or osmolality—salt concentration] is not good for the cells.”
Many researchers prefer a large environmental chamber that encloses the stage and the lenses. Luby-Phelps says a chamber that just sits on the stage is not as effective as a large environmental chamber, “because the lens is a huge heat sink—it’s a big chunk of metal. If it’s not heated to 37°[C], it will suck the heat right out of your sample, particularly if you’re using an oil immersion lens, and it will never get to 37°.”
A variety of setups exist that can be designed and made in-house, or purchased. Luby-Phelps’ system actually uses two Plexiglas boxes—a smaller one with controlled humidity and CO2 that encloses the sample (so the humidity does not affect the microscope very much) and a larger one that keeps everything at 37°C. Spector, on the other hand, prefers a single box (with doors) that snugly covers “from the bottom of the stage right up above the microscope [and] in which CO2, humidity and temperature can all be controlled.”
These days, such units are typically manufactured by third parties and purchased through the microscope vendor at the time of sale. Environmental chambers also can be obtained directly from the manufacturer to retrofit an existing system.
See the light (but not too much)
Light is a double-edged sword in live-cell imaging. It’s necessary to capture an image, but it can cause phototoxicity, bleaching and other damage. “The worst thing you can do is put your dish on a fluorescent microscope and sit there and look at a cell—you’re giving it so much light radiation that you’re probably pulling out of the cell cycle,” cautions Spector.
The key is to keep both intensity and duration of light to a minimum: Use a sensitive detector to make the most from the least amount of illumination, and only illuminate the sample when data are actually being captured by having a camera trigger the light (or vice versa).
There are essentially four types of cameras to consider, explains Rachit Mohindra, product manager at Photometrics. A basic color-imaging camera is used for photodocumentation, and it may not have the speed or sensitivity for most live-cell-imaging applications. CCDs can be very sensitive, with low noise, but have traditionally offered relatively low frame rates.
For the majority of users, Mohindra recommends a camera based on a CMOS sensor, because “they have the most ideal balance between sensitivity, frame rate and resolution … and are particularly useful in looking at very rapid events.”
The fourth type of camera, typically used in light-starved environments like super-resolution microscopy and single-molecule fluorescence, uses an electron multiplication CCD (EMCCD) sensor, which is “typically about as sensitive as you can get with a camera”—capable of converting about 95% of photons into a measured signal, says Mohindra. Such cameras are extremely temperature-sensitive (they need to be cooled down to -70°C to -80°C) and tend to cost about $30,000 to $40,000.
Data
Like many current lab technologies, so much data can be generated so fast in live-cell imaging—up to a gigabyte (GB) per second, in some cases—that managing and processing it becomes a challenge. “We recommend some pretty beefy computers to try and keep up with it, with solid-state hard drives just to be able to save the data at an appropriate rate,” says Mohindra.
Slower but longer-term experiments can be equally challenging. Luby-Phelps’ core has users acquiring 80-GB data sets overnight. And that, she says, requires powerful hardware and software to store and process.
Still interested in doing live-cell imaging? The main things to keep in mind, according to Spector, are the chamber (“just maintain the cells as close to how you grow them in an incubator”), the camera (fast and sensitive enough to allow for minimum illumination), computer, software to control the system and “using a glass-bottom dish, so your image quality is good.” Then you need to figure out how to use it all. But not to worry, live-cell imaging is becoming more routine in the cell-bio community, and there are lots of core staff members and investigators to learn from.
Image: iStockPhoto