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Jun 1, 2013 (Vol. 33, No. 11)

Lights, Camera, and Lots of Cellular Action

  • Click Image To Enlarge +
    An H9 embryonic stem cell line stably transfected with a GFP-Oct4 promoter construct 65h after seeding and continuous imaging. Time-lapse images over a total of five days allow visualization of how inhomogeneities in colony expression of Oct4 develop over time. Hundreds of colonies are imaged over ~1cm2 every 45 minutes in each experiment. [NIST]

    Innovations in live-cell imaging are reshaping scientific paradigms, opening the door to new approaches for scientific inquiry and opportunities for predictive modeling. Emerging technologies allow more in-depth molecular and cellular data collection from living cells and tissues.

    Technology advances bring new sets of challenges, including the development of stable processing environments, and analyses of vast quantities of data in robust, transferable methodologies. Live-cell imaging was one of the many technology topics discussed at CHI’s High-Content Analysis conference.

    Ammasi Periasamy, Ph.D., professor of biology and director, W.M. Keck Center for Cellular Imaging, University of Virginia, discussed what he called the “fundamentally important role” that technology development plays in the advancement of live-cell imaging.

    “Gene chips, high-throughput sequencing, and real-time manipulation of macromolecules in living cells have allowed scientists to observe and understand the central dogma that constitute cellular life,” Dr. Periasamy said.

    “Super-resolution techniques have demonstrated that the wavelength of light does not have to ultimately define the spatial resolution, and 3D localization has been demonstrated, within limited volumes, in super-resolution technology,” he added.

    Further, Dr. Periasamy said that “improvements of key fluorophore properties, particularly greater photostability, larger Stokes shifts, and smaller sizes, will help to improve current probe designs, which will expand the design space to create a new generation of smart probes. Phytochrome, phototropin, and light-sensitive ion channels derived from bacteria will be used to create tools for live-cell imaging and optogenetics in the near-term future.”

    However, despite technological developments, even when high-quality image data are collected, the analysis required to obtain robust quantitative information is extremely challenging, as is the assessment of image segmentation and tracking software accuracy, as well as the analysis of very large datasets.

    This is an exciting time for biology, according to Anne Plant, Ph.D., chief, biosystems and biomaterials division, National Institute of Standards and Technology (NIST). “Technology has evolved to where we can now probe large numbers of individual cells in the time domain by light microscopy. This capability provides insight into the statistical details of population dynamics, and tells us about the forces that constrain cell response within an environment.

  • Predictive Model Development

    “These dynamic data allow the development of predictive models, and also provide a direct correlation between individual cell response and the fate of that cell,” she continued.

    Promoter activity varies from cell to cell, and results in variations in cell fate. In addition, the rate at which promoter activity fluctuates with time in individual cells varies.

    The time-dependent fluctuations in expression levels for a transcription factor such as Oct-4 can provide a rate constant that allows for the prediction of future expression levels.

    “With stem cells, we also are examining the data for characteristic patterns of expression that might provide predictive information, or correlations, between expression level and the observed phenotype,” Dr. Plant reported.

    “We collect time-lapse image data from many colonies, with as much time resolution as we can, for the longest period possible. This allows observation of the events in stem cells that lead to spontaneous differentiation, and the characteristics of colonies that maintain their pluripotent state,” added Michael Halter, research scientist, cell systems science group, NIST. “The goal is to develop tools that will allow routine data collection and analysis at this scale for the stem cell biology community and provide insight into control of stem cell differentiation.”

    The researchers used inverted fluorescence wide-field microscopes with broad-spectrum light sources, motorized stages and filters, and environmental chambers. They also developed and evaluated automated segmentation and tracking algorithms. Drs. Plant and Halter then used specialized software tools to visualize very large image datasets.

    Drs. Plant and Halter noted that two technologies required to improve the quality of live-cell imaging data are the continuous monitoring of temperature, CO2, and O2 at the bottom of the culture vessel where the cells reside, and the reduction of background fluorescence signal present in cell culture media.

  • Utilizing the Doppler Effect

    David Nolte, Ph.D., professor of physics at Purdue University, also discussed his group’s live-cell imaging work.

    “We were investigating a full-frame imaging approach related to optical coherence tomography (OCT) using digital holography. Holography is extremely sensitive to minute motions, and we noticed highly developed speckle fluctuations in the images from living tissues,” Dr. Nolte said. “As we explored these dynamic processes, we came to understand that the origin was in the multiple types of motion occurring at the subcellular scale.”

    He noted that “when light shines on things that move, there is a frequency shift, known as the Doppler effect. This is like the effect at a train crossing—the approaching train horn is high-pitched, but as it passes by it becomes low-pitched. When we shine light on living tissues, all the internal motions of the cells cause a similar effect on the light frequency, which we detect and relate back to intracellular motions.”

    Tissue dynamic imaging (TDI) is the use of label-free and noninvasive intracellular motion as an imaging contrast agent. TDI provides an opportunity to develop a new 3D tissue screen for many types of drugs in early drug discovery.

    Intracellular motions are altered by different mechanisms of action and generate drug-response spectrograms that act as fingerprints for phenotypic profiling. A general technique, TDI can be used for a wide range of applications in biomedicine, drug discovery, biology, and in the clinic. Anywhere cells are aggregating and living, this new form of microscopy can provide unique insights.

    “No one knows how to get high-quality information out of living tissue, so they avoid asking those kinds of questions. With TDI, we now provide that needed tool, and we foresee a significant growth in live-tissue imaging,” Dr. Nolte concluded. “TDI relies on light, and we can go about 1 or 2 mm into tissue. This is much deeper than other light-based techniques, but it is not anything like magnetic resonance imaging.”


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