Tandem Dyes

The commercial availability of antibody-linked fluorescent dyes has expanded rapidly over the past decade. Where researchers were once limited to a handful of common dyes (FITC, PE, APC, PerCP, etc.), they can now choose from an extensive list of fluorescent dye lines – spanning the light spectrum from IR to UV. Additionally, researchers can now use several tandem dyes to improve the fluorescent signal for their experiments. However, while tandem dyes can serve as powerful research tools, they can also introduce a new set of problems to consider. Here we explore the benefits of tandem dyes and review some of the difficulties that they present.

Basics of Fluorescence
Fluorescence is described in terms of excitation and emission. A fluorochrome may be excited by a laser at a defined excitation wavelength, at which time the molecule absorbs light photons. Then, as it returns to its ground state, the fluorochrome emits those photons with less energy and at a longer wavelength. The difference in these two excitation and emission wavelengths is called the Stokes shift. Fluorochromes exhibiting larger Stokes shifts are generally more desirable for fluorescent research, as the emitted light can be easily distinguished from the exciting light source.

In this example image below created by FluoroFinder’s Spectra Viewer, all three dyes share the same excitation characteristics because they use the same donor, while the acceptor molecules differ. This changes the emission characteristics and causes varying degrees of Stokes Shift.

Tandem Dyes
One method for increasing the observable Stokes shift value is to use a tandem dye. Tandem dyes refer to two covalently bonded fluorochromes, a donor and an acceptor. As the donor molecule becomes excited, it transfers photons to the acceptor molecule via a radiationless process called Förster Resonance Energy Transfer (FRET). The difference between the excitation wavelength of the donor and the emission wavelength of the acceptor allows the tandem dye to display a greater Stokes shift than either individual molecule.

The Trouble with Tandems
Because the photon transfer efficiency in tandem pairs can differ each time the conjugation is performed, the resulting spectral overlap values can vary from lot to lot. Manufacturers have recently made great developments toward reproducible formulations and improved packaging consistency to reduce this lot-to-lot variation. However, each tandem dye antibody lot used in an experiment will require its compensation control to ensure that their spectral characteristics are identical (PMID: 4415368). Additionally, the covalent bonds that link tandem dyes may degrade over time and under certain conditions. For example, light exposure, temperature changes, lysing, and fixation can all affect tandem dye bond quality. Tandem dye breakdown can cause false-positive signals in the donor channel, giving the appearance of under-compensation. However, increasing compensation between channels will not resolve the problem, and may result in false negatives due to overcompensation.

While quality control of all fluorescent reagents is critical to provide reliable results, it is especially important in the case of tandem dyes. New lots and shipments of tandem dyes should always be tested for acceptable performance before use and regularly tested for stability to identify when a reagent should be removed from use.

 

Designing with Tandems
Designing a flow cytometry experiment using tandem dyes is like designing a panel with traditional dyes. Panel design tools like FluoroFinder make complex multicolor panel design easy by bringing all the available options into one place and dynamically updating those options as you select antibody/fluorochrome combinations and assign them to channels of your specific cytometer.

 

Fluorophore vs Fluorochrome?
Many researchers use the terms interchangeably, but they are not necessarily synonyms. Make sure you know the difference to accurately describe your fluorescent experiments!
A fluorochrome refers to any fluorescent dye used to stain cells or tissue for microscopic examination.

A fluorophore refers to any molecule or functional group which is capable of fluorescence.
Therefore, all fluorochromes are fluorophores, but not vice versa…

 

Available Tandem Dyes

SupplierTandem Dyeex/emSpectra Viewer Link
VariousAPC-Cy™5.5650/695View
VariousAPC-Cy™7650/785View
VariousPE-Cy™5565/667View
VariousPE-Cy™5.5565/672View
VariousPE-Cy™7565/785View
VariousPE-Texas Red®565/613View
VariousPerCP-Cy™5.5482/695View
AbcorePE-Dyomics 590488/599View
AbcorePE-Dyomics 647488/672View
BD BiosciencesBD Horizion Brilliant™ Ultraviolet 496 (BUV496)351/491View
BD BiosciencesBD Horizon Brilliant™ Ultraviolet 563 (BUV563)351/560View
BD BiosciencesBD Horizon Brilliant™ Violet 605 (BV605)407/605View
BD BiosciencesBD Horizon™ PE-CF594496/612View
BD BiosciencesBD Horizon Brilliant™ Ultraviolet 615 (BUV615)350/616View
BD BiosciencesBD Horizon Brilliant™ Violet 650 (BV650)407/650View
BD BiosciencesBD Horizon Brilliant™ Ultraviolet 661 (BUV661)351/657View
BD BiosciencesBD Horizon™ APC-R700652/704View
BD BiosciencesBD Horizon Brilliant™ Violet 711 (BV711)407/711View
BD BiosciencesBD Horizon Brilliant™ Ultraviolet 737 (BUV737)350/732View
BD BiosciencesBD Horizon Brilliant™ Violet 750 (BV750)405/745View
BD BiosciencesBD Horizon Brilliant™ Violet 786 (BV786)407/786View
BD BiosciencesBD Horizon Brilliant™ Ultraviolet 805 (BUV805)351/797View
BD BiosciencesAPC-R700652/704View
BD BiosciencesAPC-H7650/785View
BioLegendBrilliant Violet 570405/570View
BioLegendBrilliant Violet 605405/603View
BioLegendBrilliant Violet 650405/646View
BioLegendBrilliant Violet 711405/711View
BioLegendBrilliant Violet 750405/750View
BioLegendBrilliant Violet 785405/785View
BioLegendPE-Fire™ 780565/780View
BioLegendPE-Dazzle™ 594565/610View
BioLegendAPC-Fire™ 750754/787View
BiotiumRPE-CF®647T565/667View
BiotiumAPC-CF®750T650/780View
eBiosciencePE-eFluor® 610565/607View
eBiosciencePerCP-eFluor® 710480/675View
eBioscienceAPC-eFluor® 780650/780View
Miltenyi BiotecPerCP-Vio® 700482/704View
Miltenyi BiotecPE-Vio® 615565/619View
Miltenyi BiotecPE-Vio® 770565/775View
Miltenyi BiotecAPC-Vio® 770652/775View
ThermoFisher ScientificPE-Alexa Fluor® 610565/626View
ThermoFisher ScientificPE-Alexa Fluor® 647565/667View
ThermoFisher ScientificPE-Alexa Fluor® 700565/721View
ThermoFisher ScientificPE-Alexa Fluor® 750565/779View
ThermoFisher ScientificAPC-Alexa Fluor® 750650/787View
ThermoFisher ScientificPE-DyLight 594565/618View