Analyzing live events makes up, perhaps, the most fundamental element of life sciences. Live-cell imaging gives scientists a crucial tool for such studies—the ability to watch, record, and analyze biology as it unfolds. Biologists can watch cells in action, and even label specific molecules to track where they are and, sometimes, what they do. To make this all work, though, the cells must stay alive.
The basics of keeping cells alive doesn’t pose too much of a problem. Kolja Wawrowsky, director of microscopy at the biobank and translational research core at the Cedars-Sinai Medical Center, says, “I find that cell viability is not an issue if: the cells are kept at the right temperature, humidity, and CO2, and high illumination intensities, UV radiation, and toxic dyes are avoided.” Wawrowsky adds, “As long as both conditions are met, cells do not suffer in my experience.”
In fact, Wawrowsky has run studies with confocal microscopy that lasted 3.5 days, with the cells remaining viable. For that, though, he notes: “A perfusion system will be required to replenish the growth media for longer imaging times.”
How to keep cell samples alive for imaging also depends on the cells and the kind of imaging desired. As Neil Anthony, director of the integrated cellular imaging core at Emory University, says, “Know what you need and how stable you want it.” Only then can a biologist create the necessary conditions to image live cells as needed.
Details of life
Other experts agree that keeping the cells alive for imaging is not that complicated, as long as a set of parameters stays in the right range. Digging deeper into the specifics, Zdenek Svindrych, microscopy imaging specialist in the department of biochemistry and cell biology at the Geisel School of Medicine at Dartmouth, says, that the microscopist must “provide proper temperature, which is 37° Celsius, a CO2 concentration that’s typically 5%, high humidity to avoid water evaporation, and a reasonably sterile environment for long time-lapse imaging.” In part, that’s not so challenging, because commercially available stage-top incubators or heated microscope enclosures will take good care of this.
Keep in mind that some parameters can move one direction more than another and still keep cells content. Although 37°C is the target, Anthony says, “a little hotter is typically worse than a little colder.”
Still, there’s more to control. “One of the technical difficulties associated with imaging at elevated temperatures is sample-focus drift caused by gradual thermal equilibration of various microscope components and the sample itself,” Svindrych explains. “Since this can take hours, a microscope with an integrated autofocus system is indispensable.”
More than alive
So, sure, live-cell imaging requires living cells, but that’s not enough. As Svindrych describes it: “More than just keeping the cells alive, you want to keep them happy—that means the live-cell imaging conditions should not affect the very biology you are trying to study.”
Some potential fluctuations don’t even need to be much to make a difference. For instance, small amounts of water evaporating from the sample might trigger osmotic stress that impacts the cells. “Mechanical stimulation—for example, when pipetting drugs to the dish—may trigger changes in the actin cytoskeleton,” Svindrych notes. “Last but not least, the intense light used during fluorescence imaging may cause various phototoxic effects, ranging from halted cell division to mitochondrial depolarization and cell death caused by accumulated reactive oxygen species.”
Trying a different kind of microscopy cannot obviate these concerns. As Svindrych points out, these challenges exist “regardless of whether it’s widefield epifluorescence, laser scanning confocal, spinning disk, or lightsheet microscopy.”
Finding the fixes
Ultimately, biologists must realize, Svindrych explains, that “alive does not necessarily mean artefact-free!” He encourages microscopists to “consider all the various ways live-cell imaging stresses your cells, and try to minimize them, especially those that are likely to cause artefacts in your particular live-cell imaging assay.”
The range of potential artefacts to address will evolve over time. “For years and years, biologists looked at cells adhered to a slide under a coverslip,” Anthony says, “but nothing in biology takes place on a glass plane.” Consequently, an increasing number of biologists image cells in three dimensions, such as in spheroids and organoids. Those forms of live cells pose new challenges, such as maintaining the necessary conditions beneath the surface.
Many of the challenges to keeping cells alive for imaging and getting accurate results depend on a balance of technique and technology. “Most of the technical issues are easy—if expensive—to solve,” Svindrych says. “A proper stage-top incubator, heated objective lens if an oil-immersion lens is used, autofocus system, well-matched interference filter sets, and sensitive cameras are all commercially available.”
Some parts, though, remain tricky at best. Artefacts caused by phototoxicity remain one of them. The problem is that such effects cannot always be measured. “So, the general rule is to use as little light and as long of time intervals between images as possible, while the researchers can still see the biology they’re after,” Svindrych says.
In some situations, the impact of phototoxicity can be seen, at least to some extent. “Sometimes, it’s possible to gauge qualitatively that some light-induced damage is happening, but in most cases a proper phototoxicity assay is too time-consuming or inconclusive,” Svindrych explains. “But most importantly, microscope users need to be aware of all these adverse effects and plan their live-cell imaging experiment in such a way as to minimize possible artefacts, with regards to the particular cellular function they’re focusing on.”
In that focus, even the lens choice impacts the overall health of the cells in the end. “Pick the lens beforehand,” Anthony recommends. “I try to sell people on a water-immersion 40x lens instead of 60 or 100x.” He adds, “You get as much signal from the sample with less light with the 40x, and you don’t kill the sample.”
That’s job one: Don’t kill the sample. As shown here, there’s more, too. The sample must be as close to the natural conditions as possible. Only then can biologist see even more about how cells really live.