Live cell imaging is made possible by a confluence of advances in imaging, computing, microscopy, and reagent technologies, connected by a deeper understanding and appreciation for cellular processes.
Cell imaging has become one of the most exciting subcategories of biological microscopy. During the past decade, cell imaging has evolved from the study primarily of fixed cells to time-lapse photos of events in living cells to real-time videos of cellular processes.
“Working with living systems is much more indicative of what’s going on in the cellular physiology,” says Ian Clements, product manager at Applied Precision (Issaquah, WA), a GE Healthcare company. “We have reached the point where we can follow the same event in the same cell over a period of time rather than simply taking snapshots.”
Expanding visualization horizons
New imaging techniques typically develop in the fixed-sample world, where sequential still photomicrographs still dominate. Researchers apply these methods to living cells as the techniques improve and become faster and more robust. That was the case with optical superresolution (OSR) techniques, which enable imaging within a narrow window below the diffraction limit of visible light (about 300 nm) and the upper limit of electron microscopy (20 nm).
One approach to OSR, termed STED (stimulated emission depletion), was discovered at the Max Planck Institute in 1994, and commercialized by Leica (the original licensee), Nikon, and Zeiss. Other OSR methods include photoactivated localization microscopy (PALM); ground state depletion individual molecule return (GSDIM); and stochastic optical reconstruction microscopy (STORM), which was developed at Harvard University during the late 2000s.
The oldest and arguably most widely adopted OSR technique is threedimensional structured illumination microscopy (3D-SIM) — invented at the University of California, San Francisco — and was licensed to Applied Precision, which continues to improve the technique and implement it in its product line. An ultrafast version of 3D-SIM can capture videos of very rapid cellular events. Twenty-four top research institutions worldwide have adopted 3DSIM in their OSR core facilities.
Although routine laboratory instrumentation tends toward simplicity and ease of use (think MS, LC), high-end experimentation (e.g., flow cytometry, cell imaging) increasingly requires a type of “renaissance” researcher, according to Mr. Clements. Because cell imaging is so novel, investigators must confirm what they see on-screen with “molecular” assays. “Ten years ago journal papers often had 20 or more authors, each of whom played a small role. Today, scientists must wear many hats and be prepared to use several high-end instrumental methods as well as more traditional tests to confirm their results.”
Imaging in physiologic context
Researchers are looking for greater control and physiological context from their live-cell imaging assays. Cell-based assays provide enhanced content and pharmacological relevance over biochemical assays, but many applications benefit from physiological modeling beyond simply placing cells in a well plate or slide. The demand for physiologically relevant assays is driven by the costs associated with promoting new drugs from development into the clinic. “Learning about a drug’s activity in an enhanced in-vitro environment reduces those risks and costs,” says Michael Schwartz, program director at Fluxion Biosciences (South San Francisco, CA).
Fluxion’s claim to fame is a cell-imaging system, BioFlux, which conducts experiments under “controlled shear flow.” Many physiological processes are mediated by physical shear stresses found in blood vessels. According to Mr. Schwartz, studying these phenomena requires re-creating those shear stresses in a controlled manner. This can be accomplished by creating laminar flow in a channel of known and reproducible dimensions. “Many physiological processes, such as platelet aggregation and leukocyte adhesion, are profoundly different if you don’t incorporate the shear stress normally present in the physiological context. Controlled shear flow provides a more realistic outcome when viewing biological phenomenon, gene knockouts, or pharmacological modulation.”
Imaging in a box
Maintaining cell viability during complex imaging experiments requires special culture chambers that sit atop an inverted microscope and control for temperature and atmosphere. Instruments capable of multiplexed analyses tend to be quite pricey and difficult to use, according to Mike Mortillaro, president of cell-imaging specialist Bulldog-Bio (Portsmouth, NH).
The concept of designing cell imagers that worked inside the warm, humid atmospheres of glove boxes or incubators originated in Asia, where many laboratories do not use airconditioning during the summer. “A lot of their instrumentation is designed to handle high temperatures and humidity,” Mr. Mortillaro explains. “If you’re designing a microscope to fit into a space-challenged lab and it already operates well at high heat and humidity, why not put it into an incubator?”
Bulldog sells two such imaging products, the JuLi™ Smart Fluorescent Cell Analyzer and the LumaScope ™ Fluorescent Live Cell Microscope, which are compact and robust enough to withstand conditions in which cells thrive. Bulldog also produces cell counters and consumables. The JuLi design operates wirelessly inside glove boxes and can transmit cell images to mobile devices through a dedicated app (including one for the iPhone/iPad).
There are several advantages to having an imager inside the incubator. Operators can analyze cells in their experimental growth environment without worrying about viability. “And researchers do not have to park themselves next to the enclosure for hours or days at a time, removing and returning samples to the incubator. Not only is that time-consuming, but it adds manipulations and variables to the experiment,” Mortillaro says.
JuLi and LumaScope—both of which are fully functional, single-channel inverted microscopes—were designed for simplicity. With all unnecessary features stripped out and highly robust components built in, the instruments cost “less than one-fifth as much as a conventional inverted microscope and one-tenth as much as a multi-well plate cell-imaging system,” according to Mr. Mortillary. The catch is they are not suitable for high-throughput or multichannel experiments.
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