Fluorophore Selection for Spectral Flow Cytometry

A major advantage of spectral flow cytometry over traditional flow cytometry is that it offers greater flexibility for fluorophore selection. However, choosing the right combination of fluorophores remains key to ensuring meaningful results. This article suggests factors to consider when selecting fluorophores for spectral instruments and comments on how reagent choice influences data resolution.

Full spectrum detection increases flexibility for assay design

The main difference between traditional flow cytometry and spectral flow cytometry lies in how the fluorescent signals are detected. “In a conventional flow cytometer, only a limited range of the light emitted upon fluorophore excitation is detected,” explains Maria C. Jaimes, VP Scientific Commercialization, Cytek Biosciences. “This restricts the choice of fluorophores to those having peak emission at the wavelength matching the optical filters present in the system. In a spectral flow cytometer, the entire range of light emitted by a given fluorophore is captured by a continuum array of detectors. Unmixing algorithms are then used to distinguish fluorophores by their spectral signatures. As a result, spectral flow cytometry allows researchers to use any fluorophore that is excited by the lasers present in the instrument, significantly increasing flexibility when it comes to reagent selection.”

Factors to consider for fluorophore selection

When choosing fluorophores for spectral flow cytometry, there are several factors to consider. First, instrument specifications play a critical role because spectral flow cytometers are not all configured identically. “Differences in laser number, wavelength, power, and laser ordering can all influence fluorophore excitation,” reports Rodrigo Pestana Lopes, Ph.D., Associate Director, Immunology Solutions & Market Development – Global Marketing, Waters Biosciences (formerly BD Biosciences). “Likewise, variations in detector arrays, detector technologies (PMTs or APDs), optical filters, signal amplification settings, and spectral unmixing algorithms can affect how fluorophores are measured and separated. These differences impact the observed brightness of each fluorophore, the shape of its spectral signature, its similarity to other fluorophores, and the amount of spillover spread generated within the panel. As a result, fluorophore performance can vary considerably between instruments.”

Sample biology is equally important since the ultimate goal of flow cytometry is to accurately resolve biological populations and cellular states. At a minimum, researchers should understand the expected expression level (high, medium, or low), expression pattern (lineage, differentiation/maturation, or functional marker), and co-expression relationships of the markers being studied. “Matching bright fluorophores with low expressed markers and vice versa is recommended to ensure the signal from weakly expressed markers is not swamped by strongly expressed ones,” says Richard Cuthbert, Ph.D., Global Commercialization Product Manager, Flow and Antibody Business, Bio-Rad Laboratories. “At the same time, you should aim to assign fluorophores that minimize spread to the most critical markers in your panel since spillover spreading error (SSE) and unmixing-dependent spreading (UDS) both reduce the resolution of positive signals.”

Besides brightness and spread, other fluorophore performance characteristics to consider include stability, size, and spectral uniqueness. Stability is especially critical when planning longitudinal studies, while size represents a key consideration when performing intracellular staining. Small-molecule fluorophores such as AlexaFluor^® 647 or Pacific Blue™ typically produce very low background and non-specific binding in intracellular staining applications because any excess fluorophore can be easily removed through washing and centrifugation steps.

“To assess spectral uniqueness, researchers often use a metric called cosine similarity, which is available in many software packages and online reagent selection tools,” says Jaimes. “In flow cytometry, this ranges from 0 to 1, where 0 indicates that two fluorophores have distinct emission spectra and 1 indicates that they are spectrally identical. Alternatively, when evaluating an entire panel of fluorophores, rather than just a single pair, a useful summary metric is the condition number. The condition number indicates how easy it is to unmix a given combination of fluorophores based on their emission profiles. Ideally, the condition number should be lower than the total number of fluorophores in the panel.”

Another factor that should be addressed during panel design is sample autofluorescence. “If possible, you should try to avoid using dyes with spectral signatures that are similar to the autofluorescence in your sample,” cautions Cuthbert. “By including an unstained control in your experimental setup, you can generate an autofluorescence signature, which can then be used as an unmixing parameter to remove autofluorescence. This is actually another of spectral flow cytometry’s advantages over conventional flow cytometry, where autofluorescence can be a big problem.”

Additionally, you will want to identify fluorophores that exhibit consistent performance across experimental conditions and over time. “Changes in fluorophore behavior caused by fixation, storage conditions, tandem-dye degradation, experimental protocols, or instrument configuration can alter spectral signatures and affect unmixing accuracy,” reports Pestana Lopes.

Lastly, you should determine whether any of the fluorophores you intend to use has specific staining requirements. “Special considerations are often required for methanol-based buffers, which are commonly used, for example, in assays designed to detect phosphorylated proteins,” advises Jaimes. “Small-molecule fluorophores are generally more resistant to these conditions than protein-based tandem dyes.”

Improving data resolution

Proper fluorophore selection improves data resolution for spectral flow cytometry in a number of ways. “Choosing fluorophores with distinct, well-separated spectral signatures reduces unmixing error, which is essentially the result of applying a precise mathematical algorithm to imprecise biological data,” says Cuthbert. “Furthermore, selecting dyes with unique emission spectra and tight emission profiles—such as Bio-Rad’s StarBright™ Dyes—maximizes data resolution, since the more different the spectral signatures of two dyes are, the more precisely those two dyes will be unmixed. Ultimately, smart fluorophore choice lets the unmixing algorithm work with less ambiguity and choosing combinations that give less spread translates directly into cleaner, more confidently resolved populations.”

Tips for assigning fluorophores to different marker types based on brightness and spillover spread

Data resolution in spectral flow cytometry requires balancing fluorophore brightness against the spillover spread introduced by that fluorophore and its interactions with other markers in the panel. A useful approach is to first consider the biological role and expression pattern of each marker, as follows:

Lineage markers (primary receptors) are typically expressed at medium to high levels and are often used to identify major cell populations. Because these markers are generally easy to resolve, they can usually be assigned to dimmer fluorophores. In addition, fluorophores selected for lineage markers should minimize spillover into markers that provide more biologically informative measurements.

Differentiation and maturation markers (secondary receptors) are frequently expressed across a continuum ranging from negative to highly positive cells. These markers often require brighter fluorophores to accurately resolve subtle expression differences, especially between negative and low-expressing populations, while maintaining resolution across the full expression range. Fluorophores assigned to these markers should not only provide adequate brightness but should also minimize spread both into and from other differentiation and functional markers.

Functional markers (tertiary receptors) are commonly associated with activation, proliferation, migration, or other dynamic cellular states. Because they are often expressed at low levels and may change substantially in response to biological conditions, they generally require the brightest available fluorophores. These markers are particularly sensitive to spillover spread generated by highly expressed markers elsewhere in the panel, making fluorophore selection especially important.

Information provided by Rodrigo Pestana Lopes of Waters Biosciences (formerly BD Biosciences)