Flow cytometry is a technology widely exploited by both researchers and clinicians. So when the Australasian Flow Cytometry Group met in September in Melbourne, both the basic and applied sides of the technology were represented.
Based on the use of a beam of laser light, flow cytometry consists of a light source and a detector array trained at a focused stream of fluid. Particles suspended in the aqueous solution pass through the beam, scattering the light, and fluorescent labels attached to the particle may be excited into emitting light at a different wavelength. This combination of scattered and fluorescent light is picked up by the detectors, and by a computer-driven analysis of the fluctuations in brightness, it is possible to derive information describing the physical and chemical structure of the target.
“We design software that straightens out the ambiguities in raw flow cytometry data,” stated David Novo, Ph.D., president and founder of De Novo Software (www.denovosoftware.com). Although flow cytometry is a long-standing technology, over the years improvements in the instrumentation and parallel advances in computer technology have greatly augmented the analytical powers of this approach to cell characterization.
Dr. Novo described flow cytometry data as consisting of multiparameter optical or electronic measurements from a vast number of cells. This data is generally displayed as one- or two-parameter histograms. The range of these measured signals from typical populations can be limited (a factor of two to four) or quite wide, often by a factor of 104–105. The limited range signals are usually amplified with linear amplifiers and digitized (assigned to discrete bins, typically referred to as channels).
“Our software is designed so the researcher can display the data in a fashion that allows firm conclusions to be drawn,” said Dr. Novo. The goal of visualizing data of a measured parameter of a sample population is to determine the actual probability function (APF). A flow cytometry data histogram is a representation of the APF of the property being measured, and this approximation is correct as long as specific criteria are met.
First, the resolution of the histogram must be matched to the data so that the displayed histogram contains sufficient detail to accurately represent the APF. If the resolution is too low, then the details of the APF are lost, while too high a resolution will result in random noise that obscures a clear picture of the data.
Second, enough cells must be sampled to prevent minor statistical variations from being interpreted as significant differences in the frequency distribution.
In order to meet the first criterion, each peak in the histogram must span enough channels so all the features of the peak(s) can be seen. The second criterion is met by collecting enough events per channel so that the channel-to-channel variation is not dominated by statistical fluctuations.
“There is a distinct difference between simply scaling data by changing the relative positions of the axis coordinates and scaling the data by changing the histogram bin-widths,” according to Dr. Novo. “The primary motivation of these transformations is to make the data appear more visually intuitive.”
The De Novo Software data-analysis product, FCS Express, allows the investigator to interpret and analyze the data in the most parsimonious fashion available. Dr. Novo and his colleagues have aimed for ease-of-use, flexibility, and simplification of complex mathematical equations in the development of their software.
Michael Olszowy, Ph.D., global marketing program manager for Molecular Probes™ at Invitrogen (www.invitrogen.com), covered a wide range of nonantibody alternative reagents for labeling cells.
One of Invitrogen’s new product offerings is the line of Vybrant® DyeCycle™ stains, which perform as an alternative to Hoechst 33342, binding quantitatively to DNA. Such dyes are important in flow cytometry for studies on the cell cycle as they reveal the relative amount of DNA in populations of cells that may be actively passing through the cell cycle.
In contrast to dyes like propidium iodide that can only penetrate dead cells, these new dyes permeate living cells in the presence of media components and other materials. Three different dye options fluoresce in the violet (440), green (530), and orange (585) ranges. These spectral properties allow accurate cell-cycle analysis in living cells on the most common laser used in flow cytometry analysis, the blue, argon 488 nm laser.
In addition, because the Vybrant DyeCycle dyes are less toxic compared to other cell-cycle labels, including DRAQ5, the dyes can also be used in a cell sorter to separate out particular populations where the cells can be regrown and studied further, according to Invitrogen.
Moreover, with one of the dyes in particular, Vybrant DyeCycle Violet dye, stem cell side population analysis may be performed using a less toxic dye on the less toxic violet laser, reports the company.
According to Dr. Olszowy, Invitrogen has developed another product designed to facilitate the measurement of DNA synthesis in cellular populations. It is a technique that allows researchers to finally move from the long-standing technology of growing cells in the presence of radioactive thymidine, a technique first developed in the 1960s.
The use of tritiated thymidine was superseded through the application of bromodeoxyuridine (BrdU), a thymidine analog, which could be detected with an antiBrdU antibody, but only after extensive manipulation of the sample. Now Invitrogen scientists have developed a more simplified approach that takes advantage of Click Chemistry.
The term Click Chemistry was coined by Barry Sharpless, Ph.D., of Scripps Institute, to describe a series of simple reactions by which larger chemical units can be linked covalently in a nonbiologic reaction that minimizes background staining.
Using EdU, a different thymidine analog that enters into a simple azide-alkyne cycloaddition (the classic click chemistry reaction), it is possible to add a fluorescent molecule to newly synthesized DNA in situ. Because the detection reagent is a small azido group attached to a fluorochrome (total MW=843 vs the 150K antibody/fluorochrome), this low molecular weight reagent diffuses easily to native DNA to find its target. In this fashion investigators can generate data on the dynamics of cellular DNA synthesis that is far more rapid, accurate, and precise than that produced through traditional methods, according to Invitrogen.
The assay, available as Click-it™ EdU Cell Proliferation assay, requires about two hours to complete. Without denaturation steps or harsh treatments, little manipulation is required as opposed to traditional protocols such as BrdU incorporation, which can take up to one and a half days.
Invitrogen has also made a significant foray into the world of nanotechnology, exploiting Qdot nanocrystals, which are highly fluorescent, nanometer-sized, inorganic semiconductor crystals. The size of the particle determines its emission and absorption spectra because in this size range (10—20 nm), the structures come to obey quantum laws, which dictate their properties and hence their spectra. This means that it is possible to engineer a vast range of colored particles.
“The current crop of Qdot nanocrystals are comparable to the best of the organic dyes in intensity,” says Dr. Olszowy, “and the narrow emission spectra of the Qdot nanocrystals result in low cross-talk as the Qdot reagents exhibit minimal spectral overlap and there is never a problem with photobleaching.”
Beckman Coulter’s (www.beckmancoulter.com) flow cytometry products include the Quanta SC (launched in 2005) and the Quanta SC MPL (December 2006) systems.
“The Quanta SC performs flow cytometry through the use of flexible filter configurations, 3-color fluorescence, side scatter, cell size, and absolute count measurements,” said Yong Song, M.D., Ph.D., cellular analysis marketing product manager. The instrument is equipped with multiple excitation wavelengths including UV, advanced digital signaling processing, and automated color for multicolor applications. Cell sizing and counting is accomplished using the Coulter Principle, which provides volume measurements that are not affected by shape, color, or refractive index.
The Quanta SC contains a patented triangular-designed flow cell for increased stability and resolution of the sample flow stream, according to the company. For example, Quanta SC can chart the course of viral infection in insect cells for protein production, when cellular volume changes in response to viral load—an indication of successful infection. The automated sample preparation includes incubation setup and saves time while providing accurate data through the elimination of pipetting error and other operator failures, reports Dr. Song.
“Recently, we successfully completed the validation of operating both a UV arc lamp and a 488 nm laser at the same time,” Dr. Song stated. “Many applications such as simultaneous determination of several fluorescent proteins, Hoechst 33342 DNA analysis, and costaining of Qdots and other 488 nm laser excitable fluorophores can now be run on our benchtop flow cytometer.”
The Quanta SC can monitor cell surface proteins through the application of the fluorescence surface density parameter, which measures changes in the concentration of fluorescence per unit area of a cell. Its capability to accomplish ploidy analysis by the accurate measurement of nuclear volume provides information on the nuclear packing efficiency signature for differentiation of benign or carcinogenic tumors in the cancer research field.
Bill Telford, Ph.D., head of the NCI cytometry core laboratory, discussed his work in new excitation sources. “Until recently,” he said, “benchtop flow cytometers were equipped with only blue-green (488 nm) and red (633 or 635) gaseous lasers.” However, the recent introduction of solid-state lasers in virtually any color has greatly expanded the repertoire of the flow cytometer, meaning that it now possible to excite a range of different dyes simultaneously.
This technology opens the possibility of designing experiments involving as many as 14 colors simultaneously. Rather than a liquid medium such as dye lasers or a gas, which gas lasers employ, solid-state lasers use a solid consisting of an active medium of a glass or crystalline host material to which is added a dopant such as neodymium, chromium, or erbium.
Many of the new colors (such as the Tsien fruit dyes) excite poorly in the range of gaseous lasers, so the new solid-state lasers opens an array of applications. In fact many useful fluorochromes frequently used for epifluorescence microscopy were almost never used for flow cytometry until now. “Virtually any known fluorescent probe can be analyzed using existing solid-state sources,” added Dr. Telford.
These novel and economical laser sources make accessible a world of new opportunities. “Instead of modifying our applications to meet the restrictions of the instrument, we are now able to redesign our instrumentation to accommodate our applications,” explained Dr. Telford.
One novel application of the new instrumentation is lipid-lipid fluorescence resonance energy transfer (FRET). Incorporation of fluorochrome-conjugated lipids with complementary excitation and emission characteristics is a method for studying the lipid-lipid interactions and their roles in modulating signal transduction. Heretofore these studies were performed using the traditional methods of confocal microscopy and cuvette-based spectrofluorimetry.
In another application of the solid laser technology, Dr. Telford and his colleagues were able to map three distinctly different caspases (caspases 1, 8, and 3) in intact cells. This approach allows researchers to follow caspase activity in living cells and define its relationship to apoptosis.
A challenge to flow cytometry engineers is designing units that can be used under less-than-ideal circumstances for work in the field. There are numerous genetic disorders and infectious diseases that are scourges in Third World countries but can be easily and effectively diagnosed with flow cytometry. These include sickle cell anemia and its bedfellow, malaria, which are widespread throughout Africa. Additionally, tuberculosis and HIV/AIDS are frequently found as a devastating combination; these maladies are also targets of rapid flow cytometric diagnosis.
A Rugged Approach
This business model has driven scientists at Guava Technologies (www.guavatechnologies.com) to design a simplified and robust flow cytometer appropriate for the Indiana Jones set. “We strive for a machine so user-friendly that all the operator needs to do to get an answer is to walk up to it and press start,” says vp of R&D and operations, David King, Ph.D.
Guava has developed four different models, covering a range of various needs, not only in remote settings but also within the broader international and life sciences clinical communities. The EasyCyte Plus System can run all currently available assays including cell counts, viability counts, apoptosis assays, and cell-cycle assays, reports the company. The machines are equipped with solid-state lasers. According to Dr. King, gas lasers are fragile, finicky, and unable to deliver the field performance that one attains with the solid lasers.
Another important innovation is the abandonment of sheath fluid in the instruments, so samples of biological fluids can be taken directly from a 96-well plate. The technology, known as capillary flow cytometry, aspirates and pumps a sample directly into the laser beam. Without a diluent there is much less waste, and disposal problems are held to a minimum. This is especially important for HIV screening where contaminated blood products must be safely disposed.
“Our software design was intended to insulate the operator from knowing how the machine works,” Dr. King continued. “Traditionally, flow cytometry was done by pros who knew the technology inside and out. We’re aiming at producing a very simple instrument delivering complex cell analytic capabilities to nonspecialists.”
The advances in software, hardware, and new fluors discussed all come together to make the technology of flow cytometry more economical and user friendly. The advances made in solid lasers have revolutionized flow cytometry, and gaseous lasers are now consigned to the museum. This will assure that the new devices will be cheaper, simpler, and more user-friendly. Thus clinical applications such as rapid screening for precancerous conditions will become more accessible, prompt, and economical.