Adding Information Through New Modalities and Techniques
“People are moving from biochemical assays to cell-based assays, because they are closer to biology and life,” says Siegfried Sasshofer, director of marketing detection at Tecan (Groedig, Austria). To use cells as effectively as possible, scientists need ways to analyze many of them under various conditions, and that often requires a microplate reader.
Beyond just using cells, scientists also want to use them in their most natural state. “People started by doing experiments on cells and then killing them to analyze the results,” Sasshofer says, “but now people want to monitor live cell-based assays and see what happens.” That can require a microplate reader that also maintains cells in a healthy condition, which requires control of environmental oxygen and carbon dioxide, as well as temperature and humidity.
Typically, a reader analyzes a signal from the cell with a detector, such as a monochromator, which can measure fluorescence, for example. Some platforms can do even more.
Perfecting the path
Getting the best data can depend on high-quality optics. To improve data from microplates, BMG LABTECH (Ortenberg, Germany) developed its PHERAstar FSX and CLARIOstar microplate readers with free-air optical paths to the top or bottom of the plate. “This is achieved by a series of softwarecontrolled, motor-driven mirrors,” explains Tobias Pusterla, international marketing manager at BMG LABTECH. “This free-air optical path shows a significant improvement in signal-to-blank ratios when compared to readers with fiber optics—both for top and especially for bottom reads.”
For cell-based assays, the imaging perspective can make a big difference in the results. “Bottom reading offers several advantages for cell-based detection,” Pusterla says, “The light collector can be placed closer to the sample—the cell layer adherent to the bottom of the well—decreasing light dissipation.” He adds, “The interfering effect of the cell-culture medium is significantly reduced.”
Beyond looking from the optimal side, a reader must look at the right depth. “On the CLARIOstar and PHERAstar FS, free air-optical bottom reading can be coupled with an automatic optical focusing system that precisely focuses light onto the cell monolayer at the bottom of the well with a 0.1 millimeter resolution and with the capability to scan the bottom of the well with a resolution of 900 points per well,” Pusterla says.
These platforms also provide a controlled environment for the cells.
Some microplate readers also include a microscope that can image cells in multiwell plates. This provides two modalities for analysis.
Tecan’s new Spark multimode microplate reader, for instance, maintains the cell environment with an integrated incubator and detects cell signals, such as gene expression, while it monitors cell confluency in microscope mode. “The microscope mode automates and simplifies many routine tasks such as cell counting and cell culture QC,” says Sasshofer. “It helps you watch the cells continuously in real time, and you get a better idea of what’s going on in a cell.”
Making such a system work depends on advanced software. “The Spark software platform,” says Sasshofer, “looks more like a smartphone app that guides you through what you want to do.” This software comes with apps that count cells, measure cell viability, quantify DNA, and more. Users can also develop their own methods through a simple drag-and-drop interface.
Many scientists already combine conventional microplate-reader data collection with microscopy. In a 2015 BMC Microbiology article, for example, a team of scientists from the Swiss Federal Laboratories for Materials Science and Technology presented a method for measuring bacterial-cell viability. They reported, “The results gained from microscopy confirmed the data obtained with the microplate reader.” This is a typical application— using a microplate reader for throughput and a microscope for confirmation. In addition, scientists can use microscopy to learn even more about a process by seeing details that a traditional microplate reader misses.
Free in 3-D
Cells live in a three-dimensional environment. Consequently, many scientists are moving to 3-D cell-based assays. “A number of industries are really going into this full bore,” says Peter Banks, scientific director at BioTek Instruments (Winooski, VT). In oncology, for example, Banks points out that 3-D cell culture is being used to discover new medicines and to test them for toxicity. 3-D cultures also allow cosmetics companies to perform toxicity testing without using animals in the process.
To study 3-D cultures using conventional multimode reading and microscopy techniques at the same time, scientists can use BioTek’s Cytation 5 cell imaging multi-mode reader. Cytation 5 works with microplates with up to 1,536 wells, along with a variety of other labware, and provides incubation and dual reagent injectors. Plus, integrated Gen5 software manages and analyzes various assays. As Banks says, “This platform supplies unmatched flexibility of detection, which is especially useful in live-cell assays of 3-D cell cultures.”
Banks and his colleagues have demonstrated this platform as it applies to cancer research. “We have measured signal transduction pathways in scaffold-based 3-D cell cultures with our microplate reader optics, but also tumor invasion assays in a spheroid-based cellular model using microscopy, all on the Cytation 5,” Banks says. “In some experiments, the two optical paths are used in the same experiment.” He adds, “In an ovarian cancer cell model, we used the microplate reader optics to measure inhibition of cytokine secretion from SKOV- 3 cells by small molecules, then, using fluorescence microscopy, tested those treated cells to ensure that the small molecules weren’t toxic to the cells.”
If a scientist wants to use conventional microplate reading plus microscopy, some balance is needed. “Using microplate reader optics,” Banks explains, “maximum sensitivity in cellbased assays is achieved using a lot of cells, but with complete confluence in the microplate, it’s hard for image-analysis software to quantify responses from individual cells.” So a user might want only about 80 percent confluence for imaging. “That 80 percent confluence still works with a microplate reader,” Banks says. “It’s just a little less sensitive.”
Getting more information is the fundamental, ongoing advance in microplate readers. It comes from improving traditional techniques and adding new data-collecting modalities.
For additional resources on microplate readers, including useful articles and a list of manufacturers, visit www.labmanager.com/microplate-tech