Due to advances on many fronts, from microfluidics to software, flow cytometry is being simplified so that it can be more readily used by a wider range of scientists and clinicians without special expertise.
GEN recently spoke with technical representatives from a number of leading flow cytometry companies to get a sense of the latest instrument trends and to find out about the emergence of novel applications.
William Godfrey, Ph.D., manager of research application development, and James Tung, Ph.D., senior staff scientist, Beckman Coulter Life Sciences, both described a current move toward simpler, less expensive, benchtop instruments that offer robust and routine operation.
Simplification involves the creation of kits for common applications and the use of combinations of markers and fluors, algorithms, and software that take the judgment out of pattern recognition and make the interpretation of results less complex. In addition, companies are trying to provide detection of as many colors as possible in the simpler instruments. Dr. Tung noted that this goal is being helped by the arrival of new off-line analysis software to analyze the data, such as his company’s Kaluza® product.
New reagents are enhancing the ability to cover spectral overlap of multiple fluors. An example is Beckman Coulter’s VersaComp antibody capture bead kit, which can be used to set compensation for multicolor flow cytometry experiments, and will enable scientists to establish color compensation on research analyzers utilizing the same reagents found in their experiments.
Dr. Tung pointed out that a particularly hot area involves work on flow cytometry analysis of microparticles that represent membrane fragments of dying cells or cancer cells. The company’s current flow cytometry offerings include the Gallios™, a mid-range benchtop instrument that provides acquisition of data from up to 10 colors. An option available for new 561 nm laser-ready Gallios systems provides researchers with the ability to analyze a greater range of fluorescent proteins and to more efficiently excite PE/PE tandems, according to Dr. Tung.
Yellow-green excitation provided at 561 nm produces less autofluorescence in cells, increasing the signal-to-noise ratio for PE and DsRed detection, added Dr. Godfrey. He sees applications coming down the road that include the use of flow cytometry to detect minimal residual disease and to sort cells that will ultimately be placed back into patients.
At Bio-Rad, Steve Kulisch serves as the manager of the cell biology business unit, which is responsible for the company’s S3™ cell sorter. Kulisch explained that when Bio-Rad surveyed the cell sorter space about four years ago, the company found the technology was used essentially only at core labs. This was primarily due to the expense and sophistication of the instruments needed for immunologists, whose applications had dominated cell sorting work in the past.
Kulisch added that as emerging sorting applications from a wider range of scientists have increased demand, these same core labs can now have wait times of up to three weeks. But “biology never waits,” continued Kulisch, and Bio-Rad viewed this situation as an opportunity to provide a high-performance, user-friendly cell sorter to handle less complex work. It also would be readily usable by scientists, who were not necessarily experts in flow cytometry.
The result: the S3, which is a benchtop cytometer that can be used to supplement larger instruments at core facilities, or as an instrument for the individual research lab. Kulisch said the S3 is much less expensive than previous cell sorters, more compact, and comes with sophisticated software that permits automatic instrument calibration and setup with minimal training.
The S3 is equipped with one or two lasers (488 nm, 561 nm, or 640 nm) and up to four fluorescent detectors plus scatter detectors utilizing established jet-in-air technology.
“It is ideal for high-speed enrichment of cell populations expressing fluorescent proteins, high-purity isolation of rare cells labeled with fluorescent markers, and the isolation of single cells for single-cell biology,” said Kulisch.
At EMD Millipore, GEN spoke with both Bob Smith-McCollum, director of marketing for open cell analysis systems, including the Amnis® FlowSight® and ImageStreamX Mark II Imaging Flow Cytometers and the Guava EasyCyte™ Flow Cytometry System, and with Jason Whalley, director of marketing for cellular analysis. Smith-McCollum noted two important trends.
One was the provision of actual images of the cells as they move through the flow cell, which is offered by EMD Millipore’s use of CCD cameras in place of photomultiplier tubes (PMTs) in its Amnis imaging flow cytometers.
Another was the growing interest by a broad range of scientists in compact, “personal” flow cytometers that can detect one to six colors. Smith-McCollum pointed out that new applications are enabled by imaging flow cytometry on the Amnis instrument. These include observation and analysis of nuclear translocation, morphometric applications, quantitation and location of fluors, and the trafficking of markers through different cellular organelles.
Whalley emphasized the simplification and miniaturization offered by the company’s Muse™ cell analyzer. According to Whalley, Muse is an entry-level cell analyzer that characterizes cells, performs direct cell counts, and quantifies protein-expression levels.
“The platform enables researchers to have access to the analytical analysis of flow cytometry at their lab bench or in the cell culture hood without any prior experience of the technology. It is a true plug and play system,” he said.
Mike Olszowy, Ph.D., head of R&D for Life Technologies’ flow cytometry business, reported that at recent flow cytometry meetings he noticed a trend away from an engineering focus on lasers and PMTs to more of an emphasis on biological questions. He has also observed a major trend toward simpler, smaller, and less expensive instruments. This is leading, he said, to a “democratization of flow cytometry.”