COURTESY OF CLAIRE M. BROWN, DIRECTOR OF THE MCGILL UNIVERSITY LIFE SCIENCES COMPLEX IMAGING FACILITYCells are at their most natural in a warm, moist, dark environment. Put them in the spotlight on the microscope stage, and they may wither like an ingénue paralyzed by stage fright.
You can’t get rid of the spotlight if you hope to image live cells, but by carefully managing temperature, pH, and humidity, you can make your stars as comfortable as possible—ensuring their onstage chemistry and activities are the same as in the privacy of the incubator.
“Environmental control is one of the most important aspects of live-cell imaging,” says David Spector of Cold Spring Harbor Laboratory in New York, who co-edited the Laboratory’s manual on live-cell imaging. And it’s one with a high payoff, he adds—“A movie is worth a million words.”
If the temperature is off by just a couple of degrees, you aren’t looking at healthy tissues. Cell division ceases, for example, and embryo heartbeats stall. “Cells are used to a pretty controlled environment in vivo,” says Claire Brown, who runs an imaging facility at McGill University in Montreal. “If they’re not in that kind of controlled environment, you don’t know if you’re looking at artifacts.” Mammalian tissues prefer 37 °C, of course, but some control systems can also chill cells if required.
The simplest imaging chamber is a coverslip over a glass slide, separated by a rubber gasket to give the cells some space, with a heated plate or air blower to keep the cells warm. For longer-term imaging, however, cells require air with the right mix of gases—oxygen as well as carbon dioxide to mediate the bicarbonate buffer. They’ll also need humidified air if the cells aren’t enclosed in a snug chamber. If the air is dry, the media will evaporate, concentrating salts and other solutes. Some cultures may require a steady influx of fresh media, as well. A wide variety of incubators are commercially available to manage both temperature and gases. Some are small chambers that sit atop the stage, while others are large boxes that enclose the entire microscope.
Here, The Scientist brings you the scoop on cell-imaging chambers both small and large, as well as some do-it-yourself options and methods for pumping media across the sample.
Small & Cozy: Stage-Top Chambers
“It does all of the jobs that a very fine incubator will do, but on the microscope stage,” says John Rendina of the stage-top chambers made by his Wilmington, North Carolina, company, 20/20 Technology, Inc. The company’s product is just one of many options, which come in two main categories: open and closed chambers. Open chambers, such as a petri dish, make it easy to access the cells but may also allow the media to evaporate. Closed systems prevent evaporation but limit accessibility.
Best for: Short movies, up to 8 hours, says Kristen Orlowski of Carl Zeiss Microscopy, LLC, Thornwood, New York
Temperature Stability: Within 0.1–0.2 °C
• Stage incubating chambers are easy to use, notes Brown, who employs them to study cell migration.
• They require very little carbon dioxide, since the volume is small.
• Because the heat extends beyond the stage with some systems, microscope components will be warm near the objective and cooler elsewhere, creating a temperature gradient that may alter focus.
• Focus is also susceptible to drafts from the doors and windows or from room ventilation.
COURTESY OF 20/20 TECHNOLOGY, INC.Tips:
• If you are using oil- or water-immersion objectives, you must add an objective heater. Otherwise, the objective will draw heat from the sample. “The instant the oil makes contact with the coverslip, the temperature of the specimendrops 5–7 °C,” says Dan Focht, president of Bioptechs, Inc., in Butler, Pennsylvania.
• To maximize heat transfer between the heating element and the culture dish, rub Heat Sink Compound from Radio Shack ($5.99) in the interface, suggests Paul Kulesa of the Stowers Institute for Medical Research in Kansas City, Missouri.
• Some researchers store objectives in a box kept at 37 °C because of concern that repeated heating and cooling might damage them.
• To help shield the microscope from drafts, Brown recommends working with the dust cover on.
• To minimize evaporation from an open chamber, you can cover the media with mineral oil.
• 20/20’s closed INC2000 costs $9,200.
• From Bioptechs, a starter set for the closed FCS2 system—including adapter, chamber, controller, and gaskets—costs $4,400. A similar starter kit for the open-chamber Delta T costs $3,685.
• The Zeiss PM S incubator, suitable for covered chambers,
The best way to keep temperature and focus steady is to encase most of the microscope itself inside the incubator. Alexey Khodjakov, who studies mitosis and centrosome duplication at the Wadsworth Center in Albany, New York, decided to box his microscopes after he first tested an autofocus system. His films contained mysterious shifts in and out of focus that he finally traced to the lab’s air conditioning system turning on. “When you heat the whole microscope you have much less thermal drift,” Orlowski says. “That big box is actually an additional buffer.” Typically these Plexiglas boxes are available from the microscope’s maker, since they must match its dimensions. Some researchers also build or order custom versions.
Best for: Longer movies and fine processes where focal drift could become a problem
Temperature Stability: Within 0.1–0.2 °C
• Stable temperature across the entire microscope
• Resists drafts or fluctuating room temperature
• The boxes limit access to the microscope, and sample, to a few strategically placed doors.
• Warm, humid air may be bad for the microscope. Heat could break down rubber components
and dry out lubricants.
COURTESY OF CARL ZEISS MICROSCOPY
• Turn on the whole system an hour or two ahead of imaging to let the temperature equilibrate.
• To protect the microscope from humidity and to limit carbon dioxide use, keep the cells in a stage-top chamber inside the larger box. Control the temperature in the big box and manage the gases in the little one.
• The Zeiss XL S incubator costs $20,000–$25,000.
Cardboard and Curtains: DIY Solutions
If the cost of a commercial big box seems prohibitive, you can also cobble one together from inexpensive materials. Kulesa came up with a blueprint for building a microscope-encasing chamber out of cardboard, reflective insulation, Velcro, and Scotch tape, in collaboration with Steve Potter, now at the Georgia Institute of Technology in Atlanta (CSH Protocols, doi:10.1101/pdb.prot4792, 2007). The incubator heater he uses to maintain temperature is perfectly adequate for his work studying cell migration in chick and quail eggs. “We’re trying to simulate mother hen,” Kulesa says. Khodjakov has also used cardboard, but currently relies on shower curtains to enshroud some of his microscopes, with a hot air blower to maintain heat. “Amazingly, that is a very reliable system,” he says.
Best for: Use in conjunction with closed stage-top chambers (or bird eggs, in Kulesa’s case), since there is no humidity or carbon dioxide control
Temperature Stability: Kulesa’s cardboard chambers are stable within a degree or two. Khodjakov says the shower curtains hold temperature as well as a rigid box.
• Inexpensive. Khodjakov gets the curtains at the local dollar store.
• Because it’s easy to disassemble the cardboard or shove aside the curtain, you can access the entire stage and microscope, making it convenient to adjust your equipment or manipulate the sample. Khodjakov’s team performs microinjections on their specimens, from any angle they wish, by simply lifting the curtain.
• There is some temperature fluctuation, since Velcro doesn’t make a perfect seal, Kulesa says.
• Unlike many commercial chambers, there is no gas or humidity control. Khodjakov notes this means you can’t use open chambers or water immersion lenses, since the water would evaporate.
COURTESY OF JESSICA TEDDY AND PAUL KULESA OF THE STOWERS INSTITUTE FOR MEDICAL RESEARCHTips:
• Place the hot-air blower well above the microscope, Khodjakov recommends. Avoid aiming hot air directly at the stage from close range, which is sure to cause temperature fluctuation around the sample.
• With any large incubator system, be careful where you put the temperature sensor as well, Kulesa advises. It needs to be close to the cells, and away from drafts, in order to regulate the temperature.
• Kulesa’s system requires $100–$150 worth of materials, including a small heater meant for egg incubators.
• Khodjakov spent $1,500 on a high-quality air blower and about $100 on the curtain, posts, and other components.
• Kulesa’s group also built its own Plexiglas boxes, for less than $1,000.
Let it Flow: Perfusion Systems
If you truly want to model physiological conditions, you might need to add a perfusion system to your chamber, suggests Pina Colarusso, who runs a live-cell imaging facility at the University of Calgary in Canada. Cells in vivo live with a regular influx of nutrients. And some types of blood cells, for example, are used to being in a moving environment. Under static culture, they may become misshapen and express atypical proteins.
Best for: Long-term imaging, cells that require continuous media flow, or experiments where you want to quickly add a drug to cultures
• No need to open the chamber to add drugs or reagents
• Near-instantaneous mixing of added compounds
• Can sample used media and check for cellular products
• It’s more effort to set up.
• The movement of media may affect the focal plane, Spector says, particularly if you’re observing subcellular structures. If you carefully control the inflow and outflow, this shouldn’t occur, Focht says.
COURTESY OF BIOPTECHS, INC.Tips:
• Equilibrate new media to the proper temperature and atmosphere before perfusing.
• Look for systems that offer smooth laminar, not turbulent, flow across the sample, so the stream doesn’t wash the cells away, Focht recommends.
• Check with your chamber vendor as to how much shear force it can take, Colarusso suggests—you don’t want the chamber to explode.
• A perfusion pump from Bioptechs costs $1,095–$1,250.
• For $14,500 you can get a different kind of perfusion system, which recirculates a small amount of media across the sample to create flow, from ibidi LLC, headquartered in Martinsried, Germany. The price includes the pump itself as well as the apparatus and tubing to hold and transfer the media; control software; and a laptop computer.