Flow cytometry has come a long way since it was first conceived by Wolfgang Göhde at the University of Münster in 1968. Originally known as Impulscytophotometrie, it became better known after a name change ten years later. The principle is straightforward, based on the use of a laser beam of light focused on a stream of fluid to detect suspended particles.
The scattering of the light beam by the particles as well as the emission of light by fluorescent tags bound to them are picked up by detectors and the signal analyzed by the computer innards of the cytometer. As the years have passed, the technology has improved dramatically through advances in hardware and software and the development of better fluors including molecules that can be presented to living cells.
“Manufacturers have responded to the needs of researchers by developing faster systems that can measure more variables over shorter time periods,” according to Gillian Bryne, product specialist at Applied Cytometry. The improvements in instrument technology, however, have run way ahead of the software that analyzes the deluge of data pouring out the new, highly advanced cytometers. For example, rare-event analysis such as detecting tumor cells in peripheral blood in ancient cytometers with a maximum acquisition speed of only 1,000–3,000 events per second is slow and tedious.
With frequencies of one stem cell in a population of millions of circulating lymphocytes, the time required to build a sufficient store of data could be prohibitive. New, high-powered machines put this number to shame with their acquisition speed of 50,000 or more events per second. Another trend that Bryne cites is the increasing number of fluors and multiple lasers that can capture as many as 18 parameters per event simultaneously.
Doctors Albert and Vera Donnenberg of the University of Pittsburgh say that Applied Cytometry software, which uses scalable parallel processing to maximize data analysis throughput, makes the analysis of rare events routine. According to Bryne, VenturiOne software allows up to 400 files to be fully studied simultaneously in less than ten seconds. This speed is achieved through the use of patent-pending multiprocessor technology, which forms the basis of Applied Cytometry’s platform.
VenturiOne version 3 also produces enhanced reports, generated from within the software itself. Because of the system’s capability to deal with large numbers of files, the new version of VenturiOne can attack data generated from multiple well formats, Bryne says. As a standard, the new software works in background calculation mode, which means that files are automatically analyzed. This feature considerably speeds up the data analysis process, she adds. When larger data files (>100 megabytes) need to be analyzed, for example in rare-event detection, however, the background mode can be switched off. This allows files to be analyzed sequentially, thereby avoiding any delays in reporting. The rapid performance of the system allows immediate review of alternative models, identifying classifier populations and outcomes, an approach that would not be practical with conventional, slower software packages.
The analytical problems of older systems are exacerbated when multiple fluorochromes are used to address the signal-to-noise ratio problem, which arises when one attempts to distinguish rare events from background. Especially with tumor material, dying cells and normal cells within the tumor can confound the analysis, requiring additional fluorescent parameters to identify and remove these events from the analytical picture Now with the improved software and by increasing the number of fluorochromes used to detect the signal, background noise can be detected and eliminated.
The Donnenbergs extol the efficacy of VenturiOne software in their investigations of cancer-causing stem cells. “Using the old software, we were only able to look at the data in a limited way, and we experienced countless crashes,” Dr. Albert Donnenberg says. “With VenturiOne, we increased our analytical speed by a factor of ten. It was amazing.”
“Today, new desktop computers have two cores, but they are not utilized to their full extent by conventional software,” Tony Burpee, CEO of Applied Cytometry, adds. “This is because the software does not tie the cores together in a fashion that allows them to perform computing tasks simultaneously. Our software does this, which means that it can tremendously accelerate not only biological jobs such as evaluating data generated by labeling with multiple fluorchromes but also other tasks involving massive amounts of data such as drug screening.”
“By the 2000s, the easy-to-understand technology of flow cytometry had not yet translated into an easy-to-use instrument,” states Jack Ball, CCO at Accuri Cytometers. Ball and his colleagues feel that flow cytometry has always been a poor stepsister to the other, more widely accepted instrumentation such as PCR and standard light microscopy. Conventional cytometers have been consigned to core facilities due to their expense, high maintenance costs, and expensive training required for operators.
In 2004 Accuri set out to design an instrument that would overcome the drawbacks of conventional devices by improving on data quality, affordability, and software while reducing complexity. Its solution was the C6 Flow Cytometer® System, which the company believes optimizes and locks together the four interdependent components of fluidics, optics, electronics, and software.
Innovations include the use of peristaltic pumps with pulse dampeners and dynamic feedback to replace conventional syringe pumps or pressurization; a simplified and stable optical design; advanced electronics and intuitive software that does not require training classes, while fitting the needs of both novice and expert.
The practical results of the application of the C6 and CFlow® software were described in a poster presentation given by Clare Rogers, Ph.D., and her colleagues at Accuri Cytometers at the “International Congress of the Society for Analytical Cytology.”
Using 24-bit digital-signal processing, the flow cytometer is able to obtain much more accurate representation and processing of fluorescence and scatter signals. Because of the wider dynamic range and greater signal resolution of the C6, there is no need to adjust detector gain or voltage, thereby saving time, precious sample material, and obviating the problem of over- or underamplification of signals, Dr. Rogers explained. Furthermore, the zoom function of the C6 allows the user to identify and precisely focus on populations of interest that might otherwise go unnoticed, as shown by the group’s analysis of rare subpopulations such as monocyte precursors.