Image Live Cells Through Time and Space

Optimizing conditions for time-lapse fluorescence microscopy

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Fluorescence imaging of live cells is a very powerful approach to the study of dynamic cellular processes and events, yielding high spatial and temporal resolution. The availability of more stable and brighter fluorophores—organic dyes, fluorescent proteins, and nanocrystals—over the past several years has greatly expanded the toolbox for researchers exploring the frontiers of cell biology. Beyond fluorescent reagents, advances in optics, sensor technology, computing power, and refined software tools have been integrated into imaging systems that are both more powerful and straightforward to use than just a few years ago. Moreover, these imaging systems—such as the EVOS® FL Auto Imaging System—are available today for use by researchers who are not imaging specialists.

The result of these innovations has been widespread adoption of fluorescence imaging in cell-based research, where scientists can apply these new tools to areas from developmental and stem cell biology to medical research, and from drug discovery to environmental studies. It is no longer necessary to be an imaging expert to acquire high-quality images, and these imaging systems are becoming mainstays in laboratories that explore cellular processes and phenomena through an array of techniques and tools. Translational research is being enabled by a “democratization” of these sophisticated life science tools.

Live-cell imaging: Caveats and rewards

Fluorescence imaging of fixed cells is relatively straightforward (compared with live-cell imaging) and is used extensively, as it generally affords images of higher quality than those derived from live cells. Cell structure and morphology information gleaned from these fixed-cell studies can also be correlated to biological states and processes, such as cell health status or progression through developmental or disease-related pathways. Fluorescence imaging of live cells, however, is the method of choice for studying dynamic processes, and time-lapse imaging of live cells can provide unique insights into events that may unfold over hours or days (Figure 1).

Despite advances in both reagents and instrumentation, live-cell imaging remains challenging for two fundamental reasons: mammalian cells are highly sensitive to light, and they require stable environmental conditions. Utmost care must be taken to protect cells from excessive light, and to maintain stable conditions of temperature, levels of atmospheric gases (O₂ and CO₂), humidity, pH, and osmotic concentration (osmolarity) to ensure that the cells being observed remain healthy. Time-lapse imaging compounds these challenges because of the multiple light exposures and extended time out of the incubator.

Controlling light factors

Live cells are highly sensitive to light, which can cause a range of deleterious effects [1]. Of these, phototoxicity from UV light and free radicals can be a significant problem when performing fluorescence microscopy, especially time-lapse fluorescence imaging. Phototoxicity can result in cell damage and artifacts, and can complicate interpretation of results (see the Tips for minimizing phototoxicity in time-lapse fluorescence imaging box). Excess light can also destroy the fluorophores (i.e., photobleaching) being used to report on the functions or structures under study.

A number of probe- and illumination-related factors should be considered when designing time-lapse fluorescence imaging experiments. First, care should be taken to optimize labeling of cellular targets (a nucleus is far easier to label than is a low-abundance protein), and no target should be labeled excessively (Table 1). The absolute signal is less relevant than the signal-to-noise ratio (S/N). Uniform labeling is particularly challenging when mixing fluorophore categories (e.g., organic dyes, fluorescent proteins, and nanocrystals), though one useful approach is to match more brightly fluorescent probes with less abundant or more disperse targets. Excessive fluorescent labeling of cellular targets can lead to:

• Nonspecific staining with increased background signals
• Physiological artifacts and structural perturbations
• Cytotoxicity

And second, image sampling should be kept to a minimum to reduce phototoxicity and photobleaching. Cell damage must be avoided and cells maintained in a healthy state to obtain data that faithfully reflect the process under study. One advantage of LED-based illumination, such as in the EVOS® Imaging Systems, is the negligible UV light emission and near-elimination of cellular UV exposure.

Controlling environmental factors

Cell biologists are adept at culturing cells that are maintained in a cell culture incubator. However, maintaining the same conditions on a microscope stage during time-lapse imaging is far more challenging. Although mammalian cells vary widely in their tolerance of nonphysiological conditions, transformed cells lines are generally more tolerant of environmental fluctuations than are primary cells (e.g., oocytes and stem cells). Maintaining environmental conditions that are close to physiological on the microscope stage is therefore of utmost importance in time-lapse imaging experiments; in the absence of environmental control, conditions will slip out of the physiological range within a few minutes. How well the environment is controlled on the microscope stage is one of the most critical factors in successful time-lapse imaging and may, in fact, decide the success or failure of an experiment.

For instance, many cell culture media are carbonate buffered and, once they are removed from the incubator, the relatively lower CO₂ concentration of air will cause an increase in the medium pH within an hour. For cell imaging on microscope stages lacking an environmental incubator, we have developed an optically clear, physiological solution (Live Cell Imaging Solution, or LCIS) that will maintain physiological pH and osmolarity for up to 4 hours at ambient atmosphere and temperature. For longer-term imaging, particularly in a microscope-stage incubator, we offer a clear cell culture medium—Gibco® FluoroBrite™ DMEM—that is based on DMEM to help preserve cell health while also providing low background fluorescence (comparable to that of phosphate-buffered saline and 90% lower than standard phenol red–free DMEM).

A stage-top live-cell chamber: The EVOS® Onstage Incubator

For imaging over extended periods, the temperature and atmospheric conditions (gases and humidity) must be tightly maintained. Among available options, a stage-top live-cell chamber that allows control of temperature and atmospheric conditions is often the best choice. We recently introduced the EVOS® Onstage Incubator, an environmental chamber that was specifically developed as an accessory to the EVOS® FL Auto Imaging System and fits on that system’s automated X-Y stage. A small, separate control unit supplies power, gas (air, CO₂, and N₂ for hypoxia experiments), and control of humidity and temperature. Several vessel holders are available to accommodate a range of culture vessels and permit easy and convenient time-lapse imaging of cells right from the incubator.

The EVOS® Onstage Incubator controls temperature with a precision of ±0.1°C, which helps maintain cell health and reduces the risk of temperature-related biological artifacts. It also minimizes thermal contractions or expansions that may cause focus instability or drift, while the EVOS® FL Auto autofocus system helps ensure that focus is maintained during time-lapse studies when cell movement may shift the focus plane. Additional focusing options for time-lapse imaging include automatic fine and coarse focus, beacons with fixed focus points, or the use of autofocus only in the transmitted light channel to minimize light damage. The preferred option will depend on cell type, staining, duration, and other imaging parameters.

Together, the EVOS® FL Auto Imaging System and EVOS® Onstage Incubator enable precise control of temperature, humidity, and three gases for time-lapse imaging of live cells under both physiological and nonphysiological conditions. Environmental settings and image acquisition parameters are all seamlessly integrated into the EVOS® FL Auto interface, creating a high-performance inverted imaging system with unmatched flexibility, ease of use, and optics for demanding time-lapse fluorescence imaging experiments.

References

1. Schneckenburger H, Weber P, Wagner M et al. (2012) J Microsc 245:311–318.