Flow cytometry is a powerful technique that has only grown faster and more comprehensive over the years, with modern instrument configurations supporting large multicolor panels of up to a dozen or more targets. Similarly, the options for fluorophores, stains and dyes for flow cytometry have dramatically expanded, with a dizzying number of diverse options now available on the market. To help narrow down your fluorophore selection, this guide will outline key specifications and categories you should keep in mind when choosing products for your flow cytometry panels.
Excitation and Emission Spectra
Perhaps the most essential information to know about a fluorophore is its maximum excitation and emission wavelengths - these numbers will tell you which lasers, filters and other fluorophores the product should ideally be used with. The maximum excitation wavelength refers to the wavelength that will most efficiently excite the fluorophore, translating to a highest intensity emission that can be achieved.1 Thus, you’ll want to choose a fluorophore with a maximum excitation wavelength as close as possible to the emission wavelength of the laser you’ll be using it with.2
The maximum emission wavelength is the wavelength at which the output emission of the fluorophore will be the most intense. This also generally corresponds to the color of the fluorophore (i.e. the “green” in green fluorescent protein). To achieve the best resolution, you’ll want to ensure that this maximum emission wavelength is compatible with the relevant filters in use in your flow cytometry system.
For example, a fluorescein isothiocyanate (FITC) dye, with a maximum excitation wavelength around 494 nm and a maximum emission wavelength around 520 nm, would work well with a laser line at 488 nm and a 530/30 bandpass filter.3 Understanding the optics of your system is the first step toward choosing a suitable fluorophore, and many manufacturers offer selection guides to tailor your choice based on laser line and filter combinations.
Stokes Shift
Energy loss that occurs as a result of molecular vibrations during the fluorophore’s excited state means the maximum emission wavelength of the fluorophore will fall at a lower energy than the maximum excitation wavelength. The difference between these two maximums is called the Stokes shift, which is also valuable information in the selection process. Fluorophores with greater Stokes shift values have the benefit of less overlap between the exciting light and emitted light.
It can be useful to view the spectrum chart of a fluorophore in order to visualize its full range of excitation and emission spectra, the maximum intensity peaks, Stokes shift and spectral overlap areas. Additionally, comparing the spectra of different fluorophores, especially by viewing them together on the same graph using one of many available fluorescence spectra viewing tools,4 can allow you to optimize your multicolor panel by selecting fluorophores with minimal overlap and spillover risk.
Brightness
Another specification to pay attention to when perusing your fluorophore options is the brightness of the fluorophore, which is the product of its extinction coefficient (its capacity to absorb light at a given wavelength, measured in M-1 cm-1) and quantum yield (the number of photons emitted per absorbed photon, a value ranging from 0 to 1).2 Many manufacturers use a scale from 1 to 5 to rank fluorophore brightness from dimmest to brightest, however, keep in mind that one manufacturer’s “5” may be different from another’s. These ratings are useful to guide you in the right direction, but something more specific, like the extinction coefficient, can give you a better idea of the product’s brightness and help you make comparisons between different manufacturers.
In general, brighter is better when it comes to fluorophore selection, but a dimmer dye may be necessary in order to minimize spillover.5 A good rule of thumb for balancing brightness and resolution is to use brighter fluorophores for low abundance targets and dimmer fluorophores for highly-expressed targets.
A more precise way to understand the relative brightness of fluorophores when factoring in other background influences like signal intensity, electronic noise, autofluorescence and non-specific staining is by looking at stain indices of fluorophores conjugated to the same antibody and tested on the same instrument. The stain index is calculated as the difference between the mean fluorescence intensities (MFI) of the positive and negative populations divided by two times the standard deviation of the negative population.6 While stain index data may already be available for some dyes, keep in mind that this data may differ from the conditions at your own lab, and you may want to run these tests yourself down the line for the most accurate determination of relative brightness.
Photostability
Photobleaching risk is lower in flow cytometry than in applications involving longer exposures, such as fluorescence microscopy, but photostability is still an important factor to consider when choosing any fluorophore. This is especially true when you’re aiming for maximum excitation efficiency, where the risk of photobleaching is highest. Unlike maximum excitation and emission wavelengths, extinction coefficient or quantum yield, photostability is harder to nail down to a single number value, although some manufacturers have rating systems to give you a general idea of relative stability. Descriptions or visualizations of results showing a fluorophore’s fluorescence intensity over time or after repeated excitations is a plus, if they’re available.
Common Types of Fluorophores for Flow Cytometry
Many different types of fluorophores and dyes have been engineered with a range of chemistries and properties; below are a few general categories you may see while shopping for flow cytometry reagents.
Fluorescent Proteins
Since green fluorescent protein (GFP) was first cloned in the 1990s, fluorescent proteins have been an invaluable tool in countless life science applications. Genes for fluorescent proteins are fused to target genes in host cells or organisms, with the fluorescent markers permanently attached to the expressed target proteins; therefore, no additional dye needs to be added to the sample.7 Fluorescent proteins come in a very wide range of options stretching from ultraviolet to infrared,8 and can also vary widely in photostability and brightness. Some popular examples include the extremely bright tdTomato (orange), the highly photostable mCherry (red) and eGFP and its variants, with more fluorescent proteins being continually developed.
Phycobiliproteins
Phycobiliproteins are another type of fluorescent protein derived from cyanobacteria, dinoflagellates and algae. These proteins can be conjugated to many different antibodies and are useful for quantitative flow cytometry due to their large size, providing around a 1:1 protein to fluorochrome ratio during conjugation.8 These proteins also have a large Stokes shift and are extremely bright with high quantum yields, however, they are not very photostable and may require lower laser powers to prevent photobleaching. Additionally, due to their high molecular weight, they may have limited applications compared to smaller fluorophores. Examples of phycobiliproteins include R-phycoerythrin (PE), allophycocyanin (APC) and peridinin-chlorophyll-protein complex (PerCP).
Small Organic Dyes
Small organic molecule dyes are among the broadest class of flow cytometry reagents that are easily conjugated to antibodies and used for both intracellular and surface staining. Many small organic molecule dyes have been synthesized and engineered to greatly improve brightness and photostability, as well as develop unique spectral properties that expand analytical possibilities.9 The diversity and versatility of these dyes have made them a popular choice for flow cytometry and they are frequently used for large multicolor panels, leveraging the advanced capabilities of newer instruments. However, these types of dyes typically have a small Stokes shift, and can sometimes suffer a loss of signal upon fixation.
Quantum Dots
Quantum dots (Qdots) are unique fluorophores made from semiconductor nanocrystals that are virtually impervious to photobleaching and are valued for their high brightness and narrow emission spectra, reducing overlap and spillover. However, use of Qdots is relatively limited as they are not easily conjugated to a wide range of antibodies, and due to their large size and poor cell permeability they are almost always used for surface staining only. Still, the unique spectral properties of Qdots make them especially useful in some applications, such as surface staining of weakly-expressed targets and spectral flow cytometry. Qdots are typically excited by UV or violet lasers and emit in different colors based on the size of the nanocrystal.10
Polymer Dyes
Polymer dyes are another relatively new type of fluorescent dye that has helped expand the use of flow cytometry in previously untapped spectral areas. Because they are made up of polymer chains they can be easily tuned and engineered to absorb and emit at specific wavelengths by altering the length of the chain and the attached molecular subunits.8 Like Qdots, polymer dyes can absorb light at UV and violet wavelengths and emit at different colors based on the polymer construction. Polymer dyes are often extremely bright with greater photostability than phycobiliproteins, although some dyes may require buffers to prevent polymer-polymer interactions when used together. Polymer dyes are frequently used in tandem constructs and an increasing number of antibody conjugates are becoming available for these fluorophores.7
Tandem Dyes
Tandem dyes are created by coupling two different fluorophores together with a covalent bond – this allows one fluorochrome (the donor) to be excited and the other (the acceptor) to emit through the process of Förster resonance energy transfer (FRET). By allowing the excitation energy to be transferred from the donor (with a higher-energy excitation wavelength) to the acceptor (with a lower-energy emission wavelength), the tandem constructs achieve much higher Stokes shifts and expand the range of emission wavelengths that can be obtained from a single laser. In tandem dyes, the donor is typically a phycobiliprotein or polymer while the acceptor is typically a small organic dye.
As an example, if you pair a PE fluorophore, which has a maximum excitation wavelength of 565 nm and a maximum emission wavelength of 578 nm, with a cyanine 7 fluorophore that has excitation and emission maxes at 756 nm and 774 nm, respectively, the resulting tandem construct, PE-Cy7, could be excited around 565 nm and still emit light in near-IR range.
In addition to increasing Stokes shifts and allowing applications further into the IR range, tandem dyes are often very bright and can be conjugated with a wide range of antibodies. However, tandem dyes do come with challenges such as their large size as well as the risk of decoupling, which requires these reagents to be treated with special care to avoid light exposure and temperature disruptions in handling and storage.
Fluorophore Manufacturers to Consider:
- Thermo Fisher Scientific/Invitrogen
- BD Biosciences
- Bio-Rad
- Beckman Coulter Life Sciences
- Proteintech
- BioLegend
- Miltenyi Biotec
References
- "Anatomy of Fluorescence Spectra," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/fluorescence-basics/anatomy-fluorescence-spectra.html
- "Selecting the Right Fluorophores for Flow Cytometry Experiments," Article by Emma Easthope, Biocompare (2020). https://www.biocompare.com/Bench-Tips/567595-Selecting-the-Right-Fluorophores-for-Flow-Cytometry-Experiments/
- "Fluorochome/Laser Reference Poster," BD Biosciences (2020). https://www.bdbiosciences.com/content/dam/bdb/marketing-documents/Fluorochrome_Laser_Poster_2.pdf
- "Online tools for viewing fluorescence spectra," Cell & Development Biology (CDB) Microscopy Core, Perelman School of Medicine, University of Pennsylvania. https://www.med.upenn.edu/cdbmicroscopycore/spectrum-viewers.html
- Maecker, H., Trotter, J. Selecting reagents for multicolor flow cytometry with BD™ LSR II and BD FACSCanto™ systems. Nat Methods 5, an6–an7 (2008). https://doi.org/10.1038/nmeth.f.229
- "The Stain Index: What Is It and What Does It Tell You?," Blog Post, BioLegend. https://www.biolegend.com/en-us/blog/the-stain-index-what-is-it-and-what-does-it-tell-you
- "Fluorophore Fundamentals for Flow Cytometry," Webinar Presented by Jolene Bradford, Labroots (2021). https://youtu.be/vC6qjCqnHzU
- McKinnon, K. M. (2018). Flow cytometry: An overview. Current Protocols in Immunology, 120, 5.1.1– 5.1.11. doi: 10.1002/cpim.40
- "Guide to Flow Cytometry Fluorophore Selection," Article by Emma Easthope, Biocompare (2021). https://www.biocompare.com/Editorial-Articles/573651-Guide-to-Flow-Cytometry-Fluorophore-Selection/
- "Qdot Probes in Flow Cytometry," Thermo Fisher Scientific. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/antibodies-for-flow-cytometry/qdot-nanocrystals-in-flow-cytometry.html