Flow cytometry allows a variety of discrete properties, from surface and intracellular antigens to proliferation history and viability, to be queried at the individual-cell level and at very high throughput. These parameters are tagged by fluorescent staining—generally by labeled antibodies, but fluorescent proteins, nucleophilic, lipophilic and amine-reactive dyes are also commonplace. Some flow cytometers can measure only one or two dye wavelengths per cell, and the availability of discreet dyes marks the practical upper limit of other instruments.
The tried and true cache of organic fluorophores, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE) and allophycocyanin (APC), are “good enough” for most researchers who mostly look at a very few parameters at a time.
Yet there is a growing demand for dyes that will complement and enhance each other in high-content multiplex flow cytometry panels.
Here we look at recent advances that have led to a wider selection of colors, narrower excitation and emission spectra, greater Stokes shifts, more stable tandem dyes and brighter fluors.
Flow cytometry 101
A fluorescent molecule absorbs light at one wavelength and emits light at a different (generally longer, less energetic) wavelength.
In most modern research flow cytometers, a cell is interrogated by different lasers—lasing at different wavelengths—sequentially as it passes. Each laser in turn has its own set of filters and detectors (channels) capable of discriminating different wavelengths of emitted light. So, for example, an instrument with five lasers, each with five channels, can discriminate among 25 unique excitation/emission combinations (this would be termed a “25-color instrument”).
For a simple three-color experiment, it’s straightforward to choose three fluors that will each be excited by a different laser. Or, similarly, three fluors (such as quantum dots) can be chosen that will be excited by the same laser, but will each emit at a specific wavelength and thus each be picked up by its own channel.
But (returning to the 25-color flow cytometer example) to take full advantage of the instrument’s capabilities, ideally each set of five fluors is excited by one laser and no other; each of these fluors emits at a distinct wavelength that can be discriminated by a single filter; and each is sufficiently bright.
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“What we and other companies are trying to develop are dyes that are single-laser excitable,” says Brenda Karim, product manager for cell biology (focusing on flow cytometry reagents) at Bio-Rad Laboratories. Many of the original organic dyes, and even some of the newer polymer dyes, have broad emission peaks: “If you have a really broad peak, you’re not going to be able to put multiple dyes that excite off the same laser in the same series … you’re removing that space for additional fluorescent dyes.”
Coming closer
The state-of-the-art dye, Karim says, “would be very bright, very stable and multicolor-compatible—and that really translates into narrow excitation and narrow emission peaks." If you look at what is currently available on the market—the organic dyes, or even the newer polymer dyes, and the nanocrystals (known as quantum dots, or qdots)—none are able to meet all of these requirements.” Traditional organic and some polymer dyes are not suitable for highly complex applications, for example, because of their broad excitation and emission peaks. And tandem dyes are known for their stability issues.
Karim does admit, though, that the polymer-based Brilliant Violet and Brilliant UV dyes “come closer to the specs—they’re bright, and they are more multicolor-compatible. They’re definitely an improvement.” According to Rui Gardner, director of the flow cytometry core facility at Memorial Sloan Kettering Cancer Center, “The stability of these polymer dyes is still a bit of an unknown.” Clearly, additional testing and improvements need to be made before there is more widespread adoption of these products. “The excitation of the Brilliant dyes is really specific—that’s the main difference with qdots,” says David Leclerc, technical director of the University of Chicago Flow Cytometry Core facility. But the emission of some of these tends to be broader than qdots—more like organic dyes.
Some dyes with large Stokes shifts have the potential to be somewhat noisy.
To understand why, it’s important to first understand how tandem dyes work. Take, for example, the fluorophore PE, which can be excited by a blue (488-nm) laser and has a peak emission at 576 nm. When coupled to the cyanine dye Cy5, the latter (called the acceptor) absorbs the light emitted by PE and re-emits it at 666 nm. By coupling different acceptors to a donor like PE, a series of fluors excited by a single laser, but with different Stokes shifts, can be created. A similar principle can be used to create polymer-based series (although because these are proprietary, details are generally not available). But the coupling is not 100% efficient, and “the acceptor molecule can still be excited by other lasers, so in terms of spillover into other channels, it’s still a problem,” points out Gardner.
In 2012, BD Biosciences acquired Sirigen Group, the company that developed the Brilliant dyes based on Nobel prize-winning research in conductive plastics (although others play in the polymer fluor game). Together, they have developed a large portfolio of dye options available for researchers performing multiparameter flow cytometry and other fluorescence-detection analysis. BD is developing a series of Brilliant fluors to be excited by blue, yellow-green and ultraviolet lasers to complement its Brilliant Violet and Brilliant UV dyes. “So suddenly you open up a huge range of possibilities and number of parameters,” Gardner says. “It’s an exciting time, where in the very near future we will have the possibility of doing 20 [to] 30 colors on a routine basis in flow cytometry.”
Not just commercial antibodies
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These days, most commercially available antibodies can be found conjugated to the fluor of your choice, “and if the company doesn’t have the antibody you want already conjugated, they’ll just do it for you,” says Leclerc. For those instances in which, say, researchers generate an antibody in-house, kits are available to conjugate it to a dye “in essentially one step,” says Nick Gee, CEO and chief scientific officer at Innova Biosciences. Innova’s Lightening-Link kits, for example, require only 30 seconds of hands-on time, and the user doesn’t need to know any chemistry, he explains. A host of non-antibody-based fluorescent flow cytometry reagents are available, as well. Cell-permeable fluorescent probes enable the study of intracellular targets without permeabilization, points out Jack Coleman, director of biochemistry at Enzo Life Sciences. Meanwhile, genetically encoded proteins, DNA-specific dyes and amine-reactive dyes used to track proliferation or mark dead cells before they’ve been fixed, for example, are available in a variety of colors “to allow you to fit those into your panel where your antibodies aren’t,” notes Karim.
“The flow market is pushing for more lasers and more channels to increase multiplexing capabilities. Dye development is following suit by providing more choices for upcoming and existing fluorescence channels,” says Christian Dose, senior manager, research and development, at Miltenyi Biotec. “The aim is, and will be, to use the whole spectra for excitation and emission, to achieve as many options as possible.” In such a game of leapfrog, the winner is likely to be the end user.
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