Editorial Article
Monday September 21, 2009
by Caitlin Smith
One of the exciting things about new technology is the creativity that emerges as different people apply it. This is happening now with flow cytometry, a technology that is old in practice but young at heart. Flow cytometry is getting a makeover—or rather, several. Whether your research would benefit from the classic simpler-is-more-effective look, or the sexy give-me-more-colors look, or the totally out-there zebrafish look, you’re sure to find a fashion to suit you. One common denominator is the drive to make it more accessible as an everyday technique. “The accessibility of the technology is really making a difference in what people can do,” says Accuri Cytometers’ President and CEO, Jennifer Baird. “One of the challenges of the flow industry is to help users discover its power and apply it more broadly.”
Seeing more and seeing faster
Flow cytometry is usually associated with counting and sorting cells. Amnis includes the functionality of imaging with their new ImageStream-X imaging flow cytometry system. In fact, according to David Basiji, president and CEO of Amnis, “this system is still the only platform available for imaging flow cytometry on the market.” Compared to its predecessor, the new ImageStream-X is 10 times faster, has up to five lasers, gives higher-quality images (up to 60X), and is more affordable. “It is also better suited to pharma screening with an integrated autosampler for multiwell plates,” adds Basiji.
If a flow cytometry system that adds imaging doesn’t suit your fancy, FluoFarma has a high-content screening (HCS) platform that includes fully automated flow cytometry, cell culture, and data analysis. They also offer a proprietary line of recombinant fluorescent biosensors for use in HCS. “The combination of specifically engineered probes with analytical flow cytometry dramatically broadens the application field of this detection technology,” says Bruno Brisson, FluoFarma’s chief business officer, “especially when image segmentation is difficult, and when acquiring more than a few hundreds of cells per condition is required. We have demonstrated that the use of these instruments as the pivotal detection unit of versatile HCS detection platforms is possible, and that it can supplant HCS by imaging in many key applications.”
Greater sensitivity and resolution is the aim of Beckman Coulter with its Gallios™ flow cytometer, which accommodates three lasers and 10 detectors, giving up to 10 colors simultaneously. “The system was designed to have very high sensitivity, excellent dim particle resolution, and a wide dynamic range of detection,” says Richard Kendall, director of global marketing for the flow cytometry business center at Beckman Coulter. “All of which was achieved using free-space air delivery to the flow cell, as well as newly designed electronics.” Beckman’s CyAn™ flow cytometer is designed more for high-throughput screening: “This instrument combines with the continuous, rapid sampling capabilities offered by the HyperCyt Autosampler and HyperView™ Data Analysis Software to enable 384-well plates to be processed in as little as 10 minutes.”
Seeing things in new ways
Millipore’s new EasyCyte8HT builds on Guava Technologies’ microcapillary flow cell technology, adding an extra red laser. “The instrument has 6-color or 8-parameter detection capabilities,” says Jason Whalley, marketing manager for flow cytometry at Millipore. The EasyCyte8HT also has a built-in 96-well auto-sampler. The new software, InCyte, is unique in providing a visual representation of 6 targets on a sample, allowing you to look at lots of data points on a kind of heat map. “Flow cytometry is a powerful technique,” says Whalley, “but with that comes the complexity of the assay development and the data interpretation.” To help with this, Millipore has just released three assay solutions kits in cell signaling to help researchers with assay development, so that they can focus instead on their own research.
The principles of flow cytometry need not be reserved for single cells alone. Union Biomedica’s COPAS® flow cytometers analyze objects that are too large for traditional instruments. For example, says Union Biomedica’s president and CEO David Strack, “these COPAS systems were designed to accommodate small model organisms like nematodes, flies, and Zebrafish—all of which are very popular for genetics research as well as drug candidate activity and toxicology screens. Prior to COPAS, a researcher was limited to relatively small experiments because each model organism needed to be sorted by hand under a fluorescence microscope.” COPAS allows high-speed sorting based on size, optical density, and multiple fluorescence channels. Its gentle air diverter mechanism means that samples experience little reduction in viability. “Early customers told us, it is terrific to know that a specific gene is turned on or off as evidenced by a presence or absence of a fluorescent signal from each animal,” says Strack, “but it would be even better if I knew where in the animal the signal was coming from.” Now Union Biomedica’s new Profiler® lets researchers detect and record up to 8,000 data points per object. The object is then digitized into peaks and valleys of fluorescence intensity, giving an optical profile of each object to show expression.
Partec has long supplied researchers with fluorescence-based flow cytometry instruments, such as its new CyFlow® Autopreparation and Autoloading Station that handles up to 400 patient samples per day. This summer, Partec introduced a unique reagent to help developing countries combat HIV infections. Their “dry”/lyophilized mAb reagent kits are designed “for flow cytometric enumeration of CD4 T-cells and CD4 percentages among lymphocytes (CD4%) in HIV monitoring and AIDS patient follow-up,” says Roland Göhde, international project manager in the science and application department of Partec’s flow cytometry division. “For the first time, any needs for cold chain and cold storage are therefore eliminated.” This is especially important for countries where dependable electricity for cold storage may not exist.
Accuri Cytometers is trying to make flow cytometry more accessible by making portable, affordable units with automated plate features that might not have the maximum number of lasers, but as Baird says, “two lasers and four colors is more than adequate” for most every day applications of the majority of their customers. Their latest instrument, the Accuri C6, fits the bill so well that one lab recently ordered multiple instruments for a huge high-throughput chemical library screening project, which will probably look at 20 or 30 million individual chemicals, according to Accuri Chief Commercial Officer Jack Ball. Ball explains another distinguishing feature of the Accuri C6—its wide dynamic range. “We opened up the scale, so we collect a much broader range of data,” he says. “Let’s say the standard flow cytometer is looking at scales of 1 to 100, or 100 to 1000, but they can’t look at all of those at once. We set it up so that you could look at 1 to 10,000 all at the same time, but you’re able to zoom in on any point and look at it in more detail.”
Toeing the spectral limit
In contrast to Accuri, BD Biosciences’ passion is adding colors to flow cytometry and making it more complex, while simultaneously making it more accessible for people to use. BD’s newest flow cytometry system is the compact benchtop cell analyzer LSRFortessa™, which has up to five lasers, 11 different laser wavelengths, and 18 colors detected simultaneously. “Do I think we’ve reached the end of the complexity? Surprisingly, the answer is no,” says Clark Mason, director of research instrumentation and marketing for BD Biosciences. “There is a demand for more than seven lasers, and we’re working on that.” He notes that the extra lasers are needed when multiple fluorophores (up to 25) are used simultaneously. “Even though, theoretically, it’d be possible to do it with four or five lasers, if you have six or seven lasers, you can reduce the amount of spectral spillover and you can get a very clean signal that doesn’t require [electronic] compensation.”
What limits the number of lasers a cell analyzer can have? “Currently, it’s a physical limitation,” says Mason. “You can always have a bigger box if you want to get more lasers in. But I think there is going to be a theoretical limit where you start to break the laws of physics, for how many spatially separated holes you could get in a flow cell. My guess is that that’s where the constraint’s going to be.”