Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Analyzing cell biology without using labels or molecular tags may puzzle researchers learned in fluorophores and fluorescent proteins, but there's good reason to try. Traditional tools, including cell stains, dyes and recombinant fluorescent tags, can alter cellular biology in unforeseen ways, potentially compromising the results.
Today’s label-free cell assays offer less invasive analyses and a wide range of applications. The technology is used for time-lapse monitoring of live cells, drug screening, cell proliferation and migration; receptor screening of targets such as ion channels, transporters, receptor kinases and G-protein-coupled receptors; and other applications.
Here are some label-free technologies used for cell-based assays today.
Light microscopy
Collecting data through a microscope by eye may seem old-fashioned, but when your computer software can quickly detect cellular data without added labels, it becomes very high-tech. Molecular Devices’ ImageXpress Micro XLS High Content Screening System automates the collection of morphology-based data from cell-based assays for high-throughput analysis. In fact, it can perform both transmitted-light and fluorescence imaging, should your experiments call for such a comparison. “The accompanying software program analyzes both types of images by quantifying cell-morphology features, such as cell size, shape and outgrowths, and transforms this information into meaningful hits,” says Grischa Chandy, senior imaging product manager at Molecular Devices.
One challenge posed by label-free optical assays is the variations in cell type, density, morphology (i.e., some cells are flatter than others) and state (i.e., alive or fixed) that lead to difficulties in segmenting transmitted-light images. “Software segmentation of label-free images depends on being able to differentiate between low-contrast objects of interest and the background,” says Chandy. “Often the edges of cells, or structures within cells, are not sharp in label-free images.” But image-analysis software has begun to have greater success tackling this problem, providing better transmitted-light image segmentation. “It is essential for the software algorithm to be flexible enough for the scientist to be able to optimize the analysis settings to match the experimental conditions, including the plate type, cell density, media, magnification and exposure times,” Chandy adds.
Cellular impedance
A different strategy for label-free cell analysis relies on electrical impedance rather than optical imaging. The xCELLigence system (formerly distributed by Roche) featured in the RTCA series of instruments from ACEA Biosciences uses electrode arrays to monitor cells in real time.
The system works by taking advantage of the electrical interactions that occur when cells and electrodes are in close proximity. The electrodes, located on the bottom of the plate, measure electrical impedance, which changes according to the extent of contact between the plate surface and cells. As more cells attach to the electrode-embedded plate, impedance rises. The system also detects changes in cell morphology; for example, impedance changes when cells spread out or contract on the plate surface.
Such data are useful in cell-proliferation studies of adherent cells. For example, researchers can quantify changes in cell numbers over time, enabling them to measure the dynamics of the changes and the effects of different experimental conditions to which researchers might subject the cells. Treatment with different drugs or nutrients, for instance, may result in informative changes in cell proliferation, which the xCELLigence system can detect label-free.
Using xCELLigence, researchers can follow cells over extended periods, making the system valuable for studies of such long-term processes as cell invasion, migration and apoptosis. Other applications include cell adhesion, cytotoxicity and monitoring of cell health and viability.
Biosensor technology
The label-free platforms available today leverage multiple biosensor strategies. Some, including surface plasmon resonance and bio-layer interferometry, are used mostly for biochemical assays.
The Revvity EnSpire® label-free benchtop platform, which can be used for both cell-based assays and biochemical assays, relies on a resonant wavelength grating (RWG) biosensor. Cells sit in standard-sized microtiter plates, within which are embedded RWG biosensors. The EnSpire Multimode Plate Reader can “read one cell-based assay with several different technologies, [such as luminescence or fluorescence,] and label-free technology to get more insights and orthogonal confirmation from the same well,” says Volker Eckelt, portfolio director for multimode detection at Revvity. The EnSpire platform is also automation-ready, uses up to 384-well plates and is compatible with several types of robotic systems.
A key component of the EnSpire platform is Corning’s Epic® technology, which also uses RWG sensors to detect changes in the wavelengths of refracted light. In 2013, Corning released a label-free, RWG-sensor-based benchtop platform of its own, the Corning® Epic® BT (PDF). The Epic BT offers “label-free cell analysis with high temporal (3-second) and moderate spatial (90-μm) resolutions,” says Ye Fang, research director of biochemical technologies at Corning. “With its small footprint, this system can also be placed inside a typical incubator, so the assays can be performed under physiological conditions.”
A second type of biosensor used in label-free platforms is the quartz-crystal microbalance (QCM). Attana’s Cell 200™ platform (formerly distributed by Tecan) uses QCM technology to detect binding kinetics of molecules in solution as they interact with cell-surface molecules (such as transmembrane receptors, for instance). When molecules bind to the cells on the QCM surface, the crystal’s vibration frequency changes proportional to the mass of molecules bound, which is useful, for instance, for cell adhesion studies.
Although newly emerging label-free technologies are promising, they are not about to usurp today’s tried-and-true labeling methods. The straightforward answers that label-based methods can provide remain invaluable. “Label-free cell analysis is still at [an] early time of its adoption,” says Fang, who believes the technology still holds “tremendous potential” for basic research and drug development. That’s particularly true, he says, for researchers “looking for new drugs with novel mechanisms of action and new biology which [other] molecular assays missed.”
Image: Corning Epic 384-well Cell Assay Microplate