SPECTRAL FLOW CYTOMETRY

Capturing the Full Spectrum

Where spectral flow differs most significantly from conventional flow cytometry is in the optical configuration and software analysis. In conventional flow cytometry, emitted photons are funneled through a series of dichroic mirrors and band pass filters that partition the light into a narrow bandwidth for detection by a set of photomultiplier tubes (PMTs). In contrast, spectral flow uses dispersive optics, such as prisms or spectrographs, that disperse photons according to wavelength across an array of detectors. This method broadens a fluorophore’s spectral profile by capturing the entire visible and near-IR spectrum. This allows for higher resolution spectral analysis over conventional optic configurations which detect only a small portion of the emission spectra. Consequently, fluorophores with very similar emission spectra that were once impossible to separate by conventional flow cytometry methods (Fig. 1), can now be distinguished through spectral flow cytometry. Figure 3 shows how the nearly identical spectral profiles of CF®660C and CF®660R can be distinguished with spectral flow cytometry, but not with conventional flow cytometry (Fig. 2). However, it should be noted that pairing of very spectrally similar dyes like CF®660C and CF®660R is not optimal dye pairing practice and would not be recommended even for spectral flow.

Compensation Vs. Spectral Unmixing

The same flow cytometry principles of panel design and spectral spillover are applicable to spectral flow cytometry. Designing multicolor fluorescence panels requires careful consideration of fluorophores with distinct spectral profiles that can be separated by the instrument. In conventional flow, when a multiparameter panel with a dozen or more fluorophores are used, overlap of emission spectra is unavoidable. This leads to noise in channels from emissions of unintended fluorophores, an issue known as spectral spillover. To address this, single-color control samples are run with each experiment to determine the amount of spectral overlap present in each detector. In conventional flow cytometry, a mathematical method known as compensation applies these controls to subtract the overlapping spectra and isolate a fluorophore’s emission profile. This is done through a correction factor that is derived from the ratio of intended and non-intended fluorophores emitting into a channel.

In spectral flow cytometry, more complex mathematical models are required to deal with spectral spillover. This is because the instrument must distinguish between multiple fluorescent profiles across the entire visible spectrum, rather than from a few distinct channels. The process of deconvoluting fluorophore emission spectra across an array of detectors is known as spectral unmixing. This type of compensation also requires experimental reference controls in addition to noise reducing mathematical algorithms such as the least squares method. This is particularly useful for cell culture samples prone to high autofluorescence. If you are interested in learning more about spectral flow cytometry, Nolan et. al have published a helpful review in Current Protocols in Cytometry.

Validated & Published on the Cytek® Aurora

In a study published by Cytek in collaboration with Biotium, researchers acknowledge the limited number of spectrally unique fluorophores presents a challenge when designing panels that take full advantage of spectral flow cytometry technology. To overcome this challenge the study focuses on developing new flow cytometry reagents using CF® Dyes to accommodate the enhanced multiplexing capabilities of spectral flow cytometry. The authors conclude the addition of spectrally unique CF® Dyes enable support of high dimensional flow cytometry analysis of 30-40 fluorescent colors, a record in flow cytometry.

We have a growing collection of over 2000 monoclonal CF® Dye primary and secondary antibody conjugates, as well as other bioconjugates. Our Biotium Choice line of primary antibodies are developed and optimized specifically for flow cytometry and are available with a selection of widely published clones against common targets.

Also see our Mix-n-Stain™ CF® Dye Antibody Labeling Kits for rapid and efficient conjugation to one of our CF® Dyes in as little as 15 minutes. For cell viability, please view our bright and extremely stable Live-or-Dye™ Fixable Viability Stains available in 14 different colors. Live-or-Dye™ stains 510/550, 665/685, 375/600, and 615/740 were designed specifically for spectral flow.

View our flow cytometry technology page to see a full listing of probes for flow cytometry and related products.