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August 01, 2017 (Vol. 37, No. 14)

GEN Roundup: Lights! Camera! Live-Cell Imaging!

Live-Cell Imaging Needs More Than Its Star Players, It Also Needs Star Producers

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    Expert Panel

    The stars of live-cell imaging—the living cells—need pampering, just like the stars of stage and screen. If anything, the cells need more pampering. Otherwise, they may act abnormally, or even perish, during their performances.

    The problem is, cells caught in live-cell imaging’s spotlight must endure unnatural conditions while continuing to act naturally. Fortunately, live-cell imaging has several ways it can ease the burden it places on cells. It can, for example, shoot “on location.”  That is, microscopy instrumentation can be made an integral part of an incubation system. Live-cell imaging can also limit phototoxicity, the harm caused when illumination is used to excite fluorescent markers, by relying on efficient light capture, or by collecting images that are only as brilliant as necessary.

    Live-cell imaging, then, is often a balancing act. Besides trying to be sparing with light while still illuminating cellular processes, live-cell imaging may try to avoid jostling cells. Hence, the growing popularity of automated environmental controls and analytical software. Such controls can sustain cells while avoiding perturbing influences, and such software can keep track of cell movements and determine which pertain to cell function, and which may be nothing more than experimental artifacts.

    By incorporating advanced technologies, live-cell imaging may keep more of its projects on schedule and under budget, especially if high-throughput workflows are supported. It may even avoid stagey productions while embracing cinéma vérité approaches, capturing cell-level dramas such as differentiation and reprogramming, host-pathogen interactions, and the kinds of interplay unique to three-dimensional (3D) ensembles. By combining specific probes and high magnification, live-cell imaging can even delve into subcellular processes.

    To realize these visions, live-cell imaging needs more than its star players. It also needs star producers—auteurs capable of controlling all aspects of a collaborative work. That’s the conclusion reached by our panel of experts. Representing some of the leading technology providers of live-cell imaging, these experts explain what is going on behind the scenes, and how academic and commercial laboratories are catching a new wave in the characterization of cellular processes.       

  • GEN: What is the biggest obstacle that investigators try to overcome when they use live-cell imaging?

    Dr. Banks: Cellular processes are dynamic. They can last a fraction of a second or take weeks to complete, depending on how they respond to intracellular and extracellular events. Cell processes that have highly variable kinetic profiles can frustrate researchers who rely on fixed-cell workflows, which are best at capturing points in time, not spans of time. Furthermore, fixed-cell workflows require the use of different cells for each time point in a kinetic profile. Live-cell imaging allows researchers to follow these processes in both time and space in the same cells.

    Dr. Appledorn: Over the last few years, researchers working with complex cellular systems have recognized the need for more physiologically relevant data and deeper, more meaningful insight into cellular function than what is achievable with conventional endpoint assays. This has brought live-cell imaging and analysis to the forefront in both academic and commercial laboratories, where relevant phenotypic measurements of biological processes are required. The main difficulties include:

    • Maintaining an environment that is consistent throughout an assay’s time course.
    • Analyzing the images and movies that are acquired.
    • Completing experiments with sufficient throughput.
    • Finding reagents that are truly non-perturbing to the biology of the cell.

    Dr. Shumate: The significant capital investment and technical expertise required have been obstacles. Traditionally, live-cell imaging has been done on a standard inverted microscope with an acrylic enclosure or environmentally controlled stage. Etaluma offers microscopes that can withstand the temperature and high humidity inside typical cell-culture incubators and hypoxia workstations. The challenge was to provide laboratories with an economical way to eliminate reliance on core facilities or large, expensive imaging instruments. Many of our operators are graduate students and laboratory associates; imaging specialists are no longer required.

    Dr. Schneider: Biological systems are incredibly complex, dynamic, and subject to extreme variability, especially in individual cells as part of a population of cultured cells. Long-term continuous analysis with single-cell resolution is critical to understanding this functional heterogeneity and the molecular mechanisms governing cell fate.

    The challenge is creating stable, manipulatable, more in vivo-like cellular environments that provide high consistency of performance while minimizing culture artifacts and permitting continuous visual measurement. Further, while high-powered imaging platforms exist, they are quite complex and difficult to operate properly. They impose a steep learning curve and constitute a significant barrier to new user adoption.

    Dr. Boettcher: Many scientists are reluctant to try live-cell imaging because they believe that it is difficult. Although there is more variability in live-cell assays as opposed to fixed-cell assays, there is also, potentially, a richer supply of information. For example, live-cell imaging systems can support high-throughput screening and provide functional readouts such as cellular movement, morphological changes over time, or long-term toxicity data for drug discovery.

    Live-cell imaging and high-throughput screening were combined in studies performed by Daniel V. LaBarbera, Ph.D., an associate professor of drug discovery and medicinal chemistry at the University of Colorado. Using the Operetta CLS High-Content Analysis system, Dr. LaBarbera studied the mechanisms controlling the epithelial-to-mesenchymal transition in 3D colorectal cancer organoids.

    To overcome the phototoxicity that can occur with fluorophores such as MitoTracker or with nuclear stains, live-cell imaging can rely on efficient light capture, incorporating objectives with high numerical apertures and long exposures, using low amounts of excitation light from stable LED light sources. Another option is to use label-free imaging with digital phase contrast.

  • GEN: Have advances in live-cell imaging made the technology easier to use on non-mammalian cells, such as yeast or bacteria?

    Dr. Banks: Yeast and bacteria are much smaller than mammalian cells. Consequently, yeast and bacteria present certain imaging challenges. Although microbes observed with live-cell imaging readily provide details about proliferation, they may withhold information about intracellular processes, which tend to be accelerated in smaller cells.

    Intracellular imaging of cellular processes requires high-resolution microscopy and, typically, fast frame rates. Along with high resolution comes a limited field of view, which could complicate studies that attempt to generate cell-population statistics. To overcome a limited field of view, investigators may need to deploy expensive cameras or assemble composite images.

    Dr. Appledorn: The use of live-cell analysis on non-mammalian cells is not an area that I follow closely. I have noticed, however, that over the past few years, many researchers have started using fluorescently labeled microorganisms, such as Listeria monocytogenes and hepatitis C virus, to examine how non-mammalian and mammalian cells interact.

    Taking a live-cell analytical approach to answering the questions around rates of infection and coinfection in multiple cell types is absolutely required to capture the kinetics of the biology under examination. I imagine there are many other uses for innovative live-cell imaging approaches to studying biofilms, yeast behaviors, and a wide variety of microorganisms, as well as whole organisms such as zebrafish and Caenorhabditis elegans. The applications are limitless.

    Dr. Shumate: Yes, we have many customers taking advantage of the high-magnification capabilities of our instruments, which incorporate 40×, 60×, and 100× objectives. We have even been able to do automated imaging with oil-immersion objectives over limited areas.

    Our customers are looking at quorum sensing, biofilm behavior, and bacterial biosensors. We also have customers interested in applications such as algal fuel and yeast chemical intermediate production. Often the organisms in these studies grow at high humidity and elevated temperatures; [these are] adverse environments where our microscopes can reside and operate with no problems over extended periods of time.

    Dr. Schneider: Yeast and bacteria present unique challenges for live-cell imaging efforts that inform imaging-based analysis. Because these microorganisms are so small, they necessitate the use of newer super-resolution platforms for the visualization of subcellular structures. Smallness also complicates multispectral imaging, which is further hampered by the presence of the cell wall/envelope.

    To facilitate the live-cell imaging of microorganisms, genetically engineered fluorescent strains and small-molecule dyes have been developed. These tools provide alternatives to bulky antibody-based methods.

    Studies of host-pathogen interactions, biofilms, and the cell cycle require systems that are not only capable of precise delivery of media and reagents, but are also compatible with continuous, long-term culture maintenance. Specialized microfluidic-based plates with machine-driven media control, such as the CellASIC® ONIX2 platform, provide an attractive option for such studies.

    Dr. Boettcher: Live-cell imaging is usually performed in aqueous growth media to keep cells as healthy as possible. Non-mammalian cells such as yeast or bacteria are inherently smaller than mammalian cells and require high-magnification and high-resolution imaging. One of the most important advances within the field of high-content imaging is, therefore, the availability of automated water-immersion objectives.

    Water-immersion objectives enable high numerical apertures (NAs) and collect up to seven times the light collected by air objectives. Moreover, high-NA objectives provide higher resolution than low-NA objectives, providing more details from yeast or bacteria. As confocality also increases resolution, the best image quality is achieved on instruments which combine both technologies—water immersion and confocality—such as the Operetta CLS or the Opera Phenix system.

  • GEN: What innovations are coming down the road for live-cell imaging?

    Dr. Banks: In the last few years, instruments have been developed that provide automated control of reagent addition, microscopy, and environmental conditions suitable for kinetic experiments spanning seconds to weeks. This has brought live-cell imaging to the non-microscopist. New innovations will be found in image analysis and fluorescent probes. As an example, we have been working with Montana Molecular, which is developing a range of biosensors for monitoring multiplexed second messengers in real time.

    Dr. Appledorn: Although live-cell imaging and analysis has typically focused on making relatively simplistic measurements of cell morphology or cell structure, it will soon be tasked with assessing complicated life-sustaining functions in multicellular biological systems (such as spheroids, organoids, and organ-on-a-chip devices).

    Tight regulation of environmental factors, such as oxygen tension, and innovations in media formulation and cell feeding, will also be required to make the best models and the most relevant measurements. As more of these models are developed and put into use, demand for non-perturbing, live-cell reagents will rise. Reagents will be needed for generically labeling cells, as well as for measuring cellular metabolism and dissecting secondary messenger signaling pathways.

    Dr. Shumate: Two innovations are making a big impact. First, 3D models such as spheroids are being rapidly adopted, as are spheroid-holding disposable labware. When these models are subjected to microscopy, subsequent analysis typically requires the acquisition of Z stacks and the deconvolution of data. Second, physiologically relevant oxygen levels, such as hypoxia, are revealing behavior not seen in traditional cell-culture incubators.

    In addition, we think that the microscopy modes used in the research setting will migrate to the screening setting. Techniques that were performed on a few cells will be automated for parallel testing. Look for super-resolution techniques, optogenetics, the monitoring of faster signaling processes, and the use of micropipette physical interactions to move into live-cell imaging.

    Dr. Schneider: Biological systems are dynamic and highly specialized, requiring unique environmental contexts to accommodate myriad cell types and applications. Microfluidic-based methods provide the best control of microenvironments. Life is also a highly coordinated 3D system with temporary checkpoints and controls; significant advances in the ‘ease of use’ of 3D culture platforms are required for basic and applied research.

    There’s also a need for machine-based algorithmic learning to assist in deciphering the multitude of parameters in each image set. Ultimately, image analysis could be performed in real time and linked directly to manipulatable environmental control for fully automated long-term culture and analysis.

    Dr. Boettcher: We see two trends—first, the growing complexity of models (such as models that combine multiple live-cell types); second, the longer duration of live-cell experiments. Keeping cells alive and healthy during imaging over extended periods remains one of the greatest challenges.

    Stain-free imaging modes such as digital phase contrast therefore gain importance. Also, next-generation probes, surfaces, and media that minimizes light-induced damage during imaging are to be expected. Yet another trend is the development of microfluidic cell-culture devices, vessels for keeping the cell environment as physiological as possible over extended periods. Such devices could, for example, allow organ-on-chip-based experiments to run for weeks.