Preparing for Live-Cell Imaging

Live-cell imaging gives you access to active biological processes

The study of living cells using time-lapse imaging is frequently referred to as “live-cell imaging”. The time lapses between the pictures can be anywhere from milliseconds to days, depending on what processes are being observed during the experiment. By using a live-cell imaging technique, you can study the dynamic biological processes your target is involved in; this is in contrast to fixed-cell imaging, where the cellular structures are frozen at a single point in time and cellular activity is at a standstill. With live-cell imaging, kinetic processes such as enzyme activity, signal transduction, protein and receptor trafficking, and membrane recycling (endocytosis and exocytosis) can all be interrogated.

Keeping cells alive during your experiment requires planning and careful handling

In order to study active biological processes, you will need to create and maintain the conditions that keep cells functioning and relatively healthy during their time on the microscope. You will need to design your experiment to be as noninvasive as possible, since fluorescent imaging can have unwanted side effects due to illumination, which isn’t something your cells are exposed to in the incubator. Live-cell imaging requires you to both keep the cells functioning during the experiment and to be able to assess whether the experimental method is causing problems that will complicate the interpretation of your results.

Key parameters to consider before you start your live-cell imaging experiment

When planning a live-cell imaging experiment, it is critical to devise an experimental plan that includes the important considerations highlighted below.

Illumination and detection

It is always best to image your live-cell samples using as little light as possible to avoid phototoxicity. This means you will need to choose the imaging system that allows you the most control of the light source so that you can minimize the wavelength range and the number of photons illuminating your cells. You should aim to use the illumination that gives you the highest signal with the lowest level of fluorophore excitation. As you restrict the intensity of light to prevent phototoxicity, your signal will be lower, so you’ll want to do your live-cell imaging on a system that has a sensitive detector (ideally a cooled CCD camera). Live-cell imaging over longer periods of time can be challenging, since your target may move out of focus during the course of your experiment. Many microscopes have autofocusing features that can help with this. Additionally, maintaining a constant temperature in the system and keeping the volume of solution in the imaging vessel constant will help with focus drift.

Labeling

You can use fluorescent proteins to tag the protein or structure you’re interested in, and the wide variety of fluorescent proteins available has made this approach very popular and robust.

For some biological questions a smaller, membrane-permeant reagent will be your best choice. These include fluorophores that, upon excitation, brightly fluoresce only if bound to an ion (e.g., Ca²⁺). Start by selecting the brightest and most specific fluorophore so that you can use it at a relatively low concentration and still get an observable signal from your target. You should always be optimizing for conditions that allow you to use the lowest amount of light to excite your fluorophore while still generating a good signal with low background. This means optimizing your experiment for the lowest intensity and shortest exposure times possible. In some cases, particularly when you wish to image over a long period of time, it is advisable to sacrifice some resolution in exchange for healthier cells. This may mean shorter exposure times, binning, or a lower magnification. You can also choose probes closer to the red end of the fluorescence spectrum when available, since they require longer wavelengths to excite them and longer wavelengths mean less phototoxicity.

Environmental control

Many cells cannot tolerate deviations from their optimal temperature, osmolarity, pH, and humidity. Requirements will vary, of course, depending on what experimental question you are asking. For example, experiments investigating cell growth and division may have a different set of requirements than experiments involving receptor activation and calcium accumulation. Some robust immortalized cell lines will tolerate being imaged for short periods of time without any environmental control. On the other hand, for longer-term imaging studies, good results with both immortalized cells and primary cells typically require tightly controlled environmental parameters.

During short imaging experiments, you can prevent changes in osmolarity and oxygen resulting from evaporation of the imaging medium by using a large volume. For longer-term studies the temperature, pH, and humidity are usually controlled using a combination of heating units and a humidified incubation chamber that also regulates CO₂, in addition to a bicarbonate-based buffering system in the imaging medium.

 
Imaging media

There are several choices of media for imaging experiments. One option is to image in the same medium that you use to culture your cells. Most cell culture media formulations contain mixtures of inorganic minerals or salts, vitamins, amino acids, nucleic acids, sugars, lipids, and other biochemicals, to which serum is usually added. The pH-buffering system in the medium typically relies on bicarbonate and externally applied CO₂, since most cells have a long-term requirement for these for internal pH regulation. One problem associated with imaging in growth medium is that illumination can cause some of the common media components (B vitamins, proteins, phenol red) to fluoresce, leading to increased background, or to generate reactive oxygen species, harming the cells under study.

Another option is to image cells in a saline-based solution, which can provide more optical clarity but cannot support long-term cell growth. These saline-based solutions can be based on a bicarbonate buffer and externally applied CO₂, or they can contain a synthetic buffer such as HEPES to maintain physiological pH values. Saline-based imaging media are not formulated to support cell growth, and that can be a problem for some experiments that occur over longer time periods or that require higher metabolic activity and growth rates.

There are commercially available media that have been developed specifically for imaging experiments. The growth media–based solutions are intended to support growth but are formulated without components found in traditional growth media that contribute to background fluorescence. Saline-based formulations offer optical clarity and buffering, but are best used for short-term studies.