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SPECTRAL FLOW CYTOMETRY

High-Performance CF® Dyes
Our industry leading dyes demonstrate superior brightness, photostability, and solubility
Largest selection of ~30 fluorophores
Take full advantage of spectral flow multiplexing with fluorophores available from blue to near-IR
Labeled antibodies & other conjugates
Over 1000 monoclonal antibodies & highly cross-adsorbed secondary antibodies conjugated to CF® dyes
Rapid & Versatile Antibody Labeling
Mix-n-Stain™ kits are easy-to-use with minimal hands-on time & no purification required
Bright & fixable viability staining kits
Live-or-Dye™ viability stains are extremely stable dead cell stains, available in 12 colors
Flow Cytometry Accessories
Buffers, fixation & permeabilization solutions, & more

Introduction

Flow cytometry is a prolific and powerful analytical tool used for biological research and the clinic. At its core, flow cytometry enables researchers to obtain highly specific information on individual cells within a sample. Recent advancements in this field have led to the development of spectral flow cytometry, a rapidly growing technology with significantly enhanced multiplexing capabilities over conventional flow cytometry. Several companies have capitalized on spectral flow with the release of the Sony ID7000™ and the well-known Cytek® Aurora. Where conventional flow cytometer instruments can detect panels with more than a dozen fluorophores, these new spectral flow cytometers are capable of accommodating multiparametric panels with upwards of 30 different fluorophores.

How does spectral flow cytometry work?

Spectral flow cytometry shares much of the same hardware associated with conventional flow cytometry. Both systems employ standard fluidic and laser technologies that enable cell-by-cell spectral analysis. For both spectral and conventional flow cytometry methods, the process begins by delivering a sample stream through a flow chamber where cells move in a single file at a constant velocity, a process known as hydrodynamic focusing. This allows uniform and efficient excitation by a set of monochromatic lasers. Emitted photons are then detected and analyzed by a combination of optics and software to identify unique spectral emissions from fluorophore labeled biomolecules.

Figure 1. Schematic comparison of conventional and spectral detection in flow cytometry. Conventional detection uses dichroic mirrors and bandpass filters to select colors of light for detection on PMTs. Spectral detection uses gratings or prisms to disperse light across a detector array. (Figure adapted from Nolan et al. (2013))

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.

 

Figure 2. Emission spectra of CF®660C and CF®660R. Emission spectrum of CF®660C and CF®660R are indistinguishable using single excitation and emission profiles with conventional flow cytometry.

 

Figure 3. Fluorophore profiles of CF®660C and CF®660R using a 5-laser spectral flow cytometer. Spectral flow cytometry acquires the emission profiles of CF®660C and CF®660R from all available lasers across the entire spectrum from blue to near-IR, allowing the spectrally similar fluorophores to be distinguished. Data was obtained from a 5-laser Cytek® Aurora.

 Conventional Flow CytometrySpectral Flow Cytometry
OpticsDichroic mirrors/band pass filtersDispersive optics: prisms or spectrographs
MultiplexingLimited by number of detectors and filters as well as emission overlap of fluorophoresImproved spectral resolution allows for more fluorophore probes per experiment
Spectral ResolutionAcquires only a narrow emission bandwidth from a single-laser excitationAcquires the entire fluorophore spectral profile for each laser
Fluorophore SeparationCompensationSpectral unmixing

Take full advantage of spectral flow with CF® Dyes

Biotium’s CF® dyes allows you to maximize the multiplexing advantages of spectral flow by offering the largest selection of spectrally unique fluorophores, many of which are licensed by Cytek® Biosciences for use in spectral flow panels. See Figure 4 below for a smilarity matrix of CF® dyes that have been validated in spectral flow by Cytek®. In addition, CF® dyes demonstrate superior brightness, photostability, and solubility over competing fluorophore probes.

We have a growing collection of over 1000 monoclonal CF® dye primary and secondary antibody conjugates, as well as other bioconjugates. Also see our Mix-n-Stain™ CF® Dye Antibody Labeling Kits for fast and efficient conjugation to any of our CF® dyes. For cell viability, please view our bright and extremely stable Live-or-Dye™ Fixable Viability Stains available in 12 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 flow cytometry probes and related products.

Figure 4. The Similarity Index is a measure of spectral similarity on a scale from 0 to 1. Values close to 0 indicate that the full spectrum signatures of the two dyes are very different from each other, and values close to 1 indicate that the signatures are very similar to each other. When designing multiplex panels, it is important to choose unique fluorophores with minimal spectral similarity. Data was provided by Cytek® Biosciences from CD4 labeling of human PBMCs using a Cytek® Aurora flow cytometer.

 

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Mix-n-Stain™ Antibody Labeling Kits

Mix-n-Stain™ kits use revolutionary technology that dramatically simplifies antibody labeling and requires no purification. The labeling is covalent and stable. Available with your choice of 24 CF® dyes, biotin, FITC, fluorescent proteins, enzymes, or haptens.

CF® Dye Protein Labeling Kits

Everything you need to label and purify antibodies or other proteins. Choose from 19 bright & photostable CF® dyes or biotin.

CF® Dye Labeled Secondary Antibodies

Many host/target species and cross-absorption options, featuring a wide spectrum of our bright & photostable CF® dyes or biotin.

Primary Antibodies

Growing collection of more than 1000 monoclonal antibodies. Many antibodies verified in HuProt™ Array and validated in IHC and other applications. Available with bright and photostable CF® dyes, R-PE, APC, PerCP, HRP, AP, biotin, or purified. Affordable 100 uL trial sizes.