As science is becoming more interdisciplinary, the tools we use to answer questions are also crossing party lines. Case in point: flow cytometry. Once a tool only used by “real” immunologists, flow cytometry is fast becoming a method by which numerous questions can be answered, from the length of a cell’s telomeres, to the state of metabolism of a cell. It is a technique that, like microscopy, depends on the detection of fluorophores.
But what are these little molecules, how do they differ from one another, and how can we use this information to better set up our experiments?
One Quick Note
Before we get started, as an aside, the terms “fluorophores”, “fluorochromes”, and “fluorescent dyes” are all used interchangeably, so pick your favorite! Also, for the sake of simplification, I am going to use this article to highlight fluorophores that are associated with antibodies, either directly or indirectly, and not those that intercalate (i.e. 7-AAD).
Doubling Up: Tandem Dyes versus Non-tandem Fluorophores
First, let’s tackle some more jargon and define “tandem” and “non-tandem” dyes.
We know fluorescence works when photons are absorbed by a molecule that has been electronically excited, and that light energy is then released as the molecule returns to its ground state. The emitted photon has a longer wavelength and less energy than the excitation photon. The difference between the excitation and emission wavelengths is known as the “Stokes shift”. When you choose a fluorophore for flow cytometry, you want to have as large of a Stokes shift as possible, since this means that the emitted light will be reliably distinguished from the exciting light source.
To capitalize on this concept, scientists developed tandem dyes. A tandem dye (or fluorophore) is composed of two covalently attached fluorescent molecules. One serves as the donor molecule while the other serves as the acceptor. This creates a unique fluorophore that has the excitation of the donor molecule and the emission properties of the acceptor.
This is possible through the phenomenon of Förster/fluorescence resonance energy transfer (FRET), which allows one fluorophore to pass its excitation energy to a neighboring fluorophore, which then emits the photon of light. The coolest part about this is that tandem dyes actually have larger Stokes shifts compared to that of the original donor molecule.
Finally, the fluorophores that are used to create tandem dyes are actually non-tandem dyes when they don’t have a buddy to hang out with. So when it comes to naming the tandem dyes, just name the two non-tandem dyes that you are using as donor and acceptor, and there you have it (i.e. PE and Cy7 are used to make the tandem dye PE-Cy7)!
While tandem dyes are incredibly important and useful for flow cytometry they are not without limitations. The biggest and most well-documented issue is that tandem dyes are extremely sensitive and degrade easily. This leads to the loss of emission from the acceptor and increased emission by the donor and is especially problematic if you’re using fluorophores that are excited by the same laser.
For instance, if you are using PE and PE-Cy7 in your panel and the PE-Cy7 fluorophore begins to degrade, the light it emits will be detected in the PE channel, thus giving you false data. One of the main causes of tandem dye degradation (aside from oxygen radicals) is exposure to light – which is totally preventable! So when you’re staining your cells, avoid exposure to light and extreme variations in temperature. The reason for the latter suggestion is that certain tandem dyes may be susceptible to cell-mediated decoupling, so slowing down cell metabolism at low temperatures can help preserve stability.
Pick Your Color
Below are some of the most well-known fluorophores used in flow cytometry. These guys are tried and true, and the core of most staining panels.
One of the brightest fluorophores
Functions to transfer light energy to chlorophyll during photosynthesis in vivo
Tandem conjugate where PE is coupled to Texas Red
Also known as ECD (Electron Coupled Dye)
Has 6 phycocyanobilin chromophores per molecule, which are similar to phycoerythrobilin, the chromophore in PE
In vivo, is an accessory photosynthetic pigment found in blue-green algae
Allophycocyanin – Cy7
Tandem conjugate where APC is coupled to a cyanine dye (Cy7)
Longest wavelength emission in the family of fluorophores excited by the red laser
Alexa Fluor 488
Incredibly stable, so very popular dye
Spectrum almost identical to FITC
Most commonly used dye for FCM
Very sensitive to pH extremes
Peridinin chlorophyll protein-Cy5.5
Tandem conjugate where PerCP is coupled to cyanine dye (Cy5.5)
Constructed using the 35 kD fluorescent protein from the dinoflagellate, glenodinium
Tandem conjugate where PE is couple to a cyan dye (Cy5)
Extremely sensitive to fixation in paraformaldehyde, so should limit exposure to no more than 4h
Based on the 6,8-difluoro-7-hydroxycoumarin fluorophore
Strongly fluorescent, even at neutral pH
Alexa Fluor 700
Far red dye, so can use in conjunction with APC and APC-Cy7
My favorite fluorophore ☺
The science of fluorophores is changing every day, with more and more dyes becoming available for use. This not only allows for larger panels, but for more reliable distinction between fluorophores detected.
Let me know what I missed, what you’d like more information on, or what your favorite fluorophore is!
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