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.
The ability to study and image live cells enables researchers to gather more relevant information vis- à -vis longitudinal cell-cell interactions, regulatory events and developmental processes, in turn providing a deeper understanding of cellular pathways. Many distinct elements go into successfully capturing images over time—whether to compare distinct time points or create time-lapse movies—from microscope optics to tracking software and hardware. Yet among the most important and often overlooked is the ability to stabilize and control the environment, not only to keep the cells happy for the duration of the experiment but also to allow the instrumentation to function at optimal performance.
Researchers generally try to reproduce the physiological conditions of the cells and tissues under study, at least to the extent that those parameters are thought to matter and it’s feasible to do so. Typically, this involves keeping them in a temperature-, humidity- and gas-regulated incubator; removing them for only brief periods when they need to be manipulated or imaged; and returning them to the incubator as soon as possible. Yet such a strategy is not workable when the imaging is to take place over many hours or days (or even weeks) without perturbing the sample.
There are several ways to create incubator-like conditions under which cells can be imaged over time. The most popular of these involves surrounding the microscope with an environmentally controlled enclosure and creating a microenvironment that sits on the microscope stage. Variations, permutations and combinations of this strategy exist both as commercial and homebrew solutions; they include a stage-top microfluidic system, automated microscope-incubator combinations and an automated microscope that’s housed in a standard incubator. Here we provide a brief overview of some of the environmental imaging options available to researchers interested in studying cellular regulatory events.
Box it up
It’s not uncommon to walk into a microscopy lab and see a big, commercially fabricated Plexiglas box surrounding everything but the eyepieces on an expensive inverted microscope.
Such boxes—often described as cages, enclosures or jackets—are generally equipped with a precisely controlled heating system that makes it easy to keep both the sample and the microscope at 37°C and relatively isolated from the lab’s ambient temperature. This is important because temperature changes cause expansion or contraction of the microscope’s metal (such as the stage or the objective) and can lead to a significant change in focus—a phenomenon called “focal drift.”
“The jacket gives excellent temperature control over the entire system, because you’re not just heating the air around the cells, you’re heating the entire metal hardware,” says Vytas Bindokas, director of University of Chicago’s Integrated Light Microscopy Core Facility. They are slow to equilibrate, but “you get the least focal drift.”
Another advantage of these types of setups is that reagents and other equipment, such as a micromanipulator or a peristaltic pump, also can be held at temperature.
In some enclosures, atmospheric gases—especially CO2, but sometimes also O2 and N2—can be regulated. It becomes trickier when it comes to humidity, which often must be enhanced to prevent evaporation and therefore changes the osmolality of cell-culture media. Yet high humidity (and resulting condensation) can be detrimental to the microscope’s optics and electronics, and in general should be avoided. Thus the major enclosure manufacturers offer small, stage-top chambers for use in conjunction with the larger heated enclosure—in which the atmosphere, including humidity, can be tightly controlled, explains Barney Boyce, co-owner of InVivo Scientific.
Enclosures are big, bulky and difficult to remove. Thus many come with doors or even a removable panel so users and service personnel can fully access the scope, points out Luca Lanzaro, CEO of Okolab.
On stage
With a microscope stage-top incubator—essentially the same as a gas chamber with the additional ability to control temperature—“it’s easier to share the microscope with others,” Lanzaro explains. “You can just lift it away and put it aside, whereas if you have an enclosure, you’re dedicating that scope to live-cell imaging.”
Lanzaro says he has seen no difference in performance between Okolab’s enclosure-plus-gas-chamber setup vs. the company’s stage-top incubator—at least when using objectives that don’t contact the coverslip. With oil- or water-immersion lenses (the norm for higher-resolution imaging) the objective acts like a heat sink, necessitating the use of an objective heater when the microscope is not in an enclosure.
In addition to stage-top incubator boxes, which with the appropriate accessories can be used with a variety of labware, there are other, more compact solutions to keep cells happy on the stage. “We contend that in order to control a microenvironment, you want to keep your environment small,” says Daniel Focht, founder and CEO of Bioptechs. The company’s Delta T system, for example, uses a disposable 35-mm coverslip bottomed dish with an optically transparent, electrically conductive coating on the bottom surface to “give uniform temperature control across the field,” Focht says. “The heated lid provides a condensate-free area so that light from the condenser remains undisturbed at the specimen plane and a gas port to introduce a controlled atomosphere.” A variety of other accessories are available, as well, including a humidification device to supply near 100% humidity to the small space above the cells and a coverglass lid that is in contact with the media from above to enable consistent contrast throughout time-lapse imaging.
MilliporeSigma’s CellASIC® ONIX is a stage-top microfluidic system that enables researchers to image cells in micro-sized cell-culture chambers while the cells are being fed. So “if you want to add a drug or put in an inhibitor, you can do that in real time without having to manipulate the sample itself,” explains Philip Lee, head of the company’s Cell Culture Systems Marketing. “In our current plates, we enable four to six different solutions that can be cycled or switched. It’s all computer programmed, so you can have any time period or sequence you want.”
Several other companies, such as Tokai Hit, Ibidi, Warner Instruments, Pecon and AutoMate Scientific, offer their own collections of stage-top accessories for observing and experimenting on cultures during live-cell imaging.
Integration
Most enclosures and stage-top incubators are manufactured by third parties and are sometimes designed for use with a specific microscope. An exception is Thermo Fisher Scientific’s EVOS Onstage Incubator which, as part of the EVOS FL Auto Imaging System, is one of the few fully integrated systems in which the stage-top incubator is controlled from within the microscope user interface, says senior product manager Magnus Persmark. “That allows for more direct and streamlined control of both image acquisition and the environmental controls.”
There are also automated imaging systems in which a microscope is part of an incubator, such as Olympus’ VivaView™ and Nikon’s BioStation platforms. And Essen BioScience’s IncuCyte® ZOOM System is essentially an automated microscope system that sits in an incubator. This “enables observation and quantification of cell behavior over time by automatically gathering and analyzing images around the clock,” according to the company’s website. In Bindokas’ opinion, “these are all true cell-culture-optimized solutions for long-term imaging studies.” Each uses wide-field optics, though, “so if you’re interested in higher resolution, using confocal, structured illumination or something like that, you’re out of luck.”
But with the availability of Plexiglas enclosures, stage-top incubators, laser focal tracking, objective heaters and perfusion, gas and humidity controls, there are plenty of workarounds for traditional inverted microscopes. “It’s possible to get good, stable imaging basically in all cases,” says Bindokas. “It just takes a little bit of work.”