The different Personalities of Fluorophores (no one is perfect)

One of my pet peeves in marketing is how fluorophores are named. It gives this illusion that all members of that named family are equal and not still defined by their individual strengths and weaknesses. 

I’ve been lecturing on fluorescent chemistry and chemical probes for almost 20 years (and I’ve also named a lot of those families I complain about)! However, one consistency over the years is how little the average biologist thinks about how their reagents are actually working and thus lumps them all together in one functional basket.

Let’s start with simple organic dyes, what makes them special and also individually annoying. We’ll compare and contrast different fluorescent technologies in a different blog.

The physical characteristics of an organic fluor that affect their utility are:

• Polarity

• Overall charge and distribution of charge

• Size

First, though, I need to get some terms out of the way.

The first is Extinction Coefficient (EC) which is a value that describes the total absorptive capacity of a fluorophore. As a general rule, the larger a fluor, the more double carbon bonds the conjugated aromatic structure contains and thus the higher absorptive capacity of the molecule. There are also some simple modification like siliconizing the fluor that will change the EC. However, a molecule can absorb all the energy in the world and still not be fluorescent. It has to be able to take the energy it absorbs and efficiently convert it to emitted photons.

Efficiency is the key there, which is called quantum efficiency or quantum yield (QY). Quantum efficiency is very labile, influenced by the microenvironment of the fluor and can also be “tweaked” by any modification to the structure that will increase its rigidity. When you see a manufacturer list the QY of a fluorescent probe, you need to ask them in what solvent the value was determined (usually AcN or MeOH) and whether it will still be a comparable performance in an aqueous media. The brightest flours would ideally have a high extinction coefficient and high quantum efficiency. Sadly, though, this not normally the relationship between size and brightness.

So, back to the physical characteristics. As an example, let’s compare and contrast two branded families of fluorophores, the Bodipy and Alexa Fluor families, both from Molecular Probes/ Life Technologies. Bodipy dyes are characterized by neutral polarity and smaller size. That may seem simple but it translates to use in distinct applications. Bodipy dyes would be the preferred dye to label lipids due to its neutral polarity. For example, if the application is to  label a phospholipid or lipid raft to look at membrane dynamics, Bodipy dyes would be at their highest quantum efficiency when nestled into the membrane and would least interfere with molecular function in such a surface. An odd side-effect, though, is also that if the compartment is heavily concentrating the probe, it will increase the total hydrophobic force of the compartment than the unlabeled probe alone. 

If the application were to label an antibody or a protein that will be suspended and imaged in an aqueous environment, Bodipy quantum yield would become less efficient and the antibody would not likely be able to sustain the same degree of labeling (fluorophore:protein ratio). Another undesirable characteristic of conjugating an antibody with a non-polar fluor is that the conjugation chemistry most often involves occupying a charged primary amine side chain of the protein (like a lysine residue). As a result, proteins and antibodies are more likely to precipitate out of solution. Alexa Fluors and some  equivalent DY dyes from Dyomics on the other hand are very desirable for protein conjugation to maintain solubility.  Although the conjugation chemistry remains the same in neutralizing the primary amine, these fluors are sulfonated conferring additional polarity to the fluorophore. My business brain wants to add that the patent covering the sulfonation of fluors like Alexa 488 expired in 2019, which leaves this area open for further development. The rest of the sulfonated Alexa Fluors will come off patent in 2021.

However, I started this blog with not all fluors are created equally, even when they share a family name and a dominant physical characteristic like polarity. For this example we can stick to the Alexa Fluors. The smallest Alexa Fluor is Alexa Fluor 350 which is a sulfonated coumarin derivative. It has an EC around 30K. Even if it had a 100% QY, it inherently can’t absorb enough energy to be independently bright. Another variable in our application of blue dyes (that I’m not going to go deep into in this blog) is that eyes, PMTs and cameras are also inefficient at  detecting such a short high energy wavelength. Listening to a talk at the AQLM course in Woods Hole one year, the instructor  stated that blue dyes are optimal for super-resolution imaging because their short wavelength confers a tighter diameter of airy disc, which in theory means that two individual points of light can be closer to one another than green or red-emitting dyes and still detect them as two individual sources of light

Sadly, blue dyes like Alexa Fluor 350 are so dim and can’t be amplified sufficiently to be individually useful for super-res in application. Their dimness makes their photobleaching rate even more exacerbated. Also, in another blog I covered sources of autofluorescence which also is prohibitive to getting a “bright” good S:N in the blue emission range. This is one of the reason the Brilliant Violet organic polymers were so important. Being able to multimerize an individual violet/blue fluor increased the total signal strength.

Many people will say Alexa Fluor 488 is akin to a gold standard fluor in pretty much any cell-based assay, whether is be flow cytometry, FRET assays, most any microscopy platforms. Small size, high solubility, high quantum efficiency and a decent extinction coefficient. However, as the molecules get bigger, like Alexa Fluor 594/ Texas Red or Alexa 647/Cy5, the individual fluorophores become larger and less soluble both of which prohibit a very high degree of labeling with the protein/antibody. Also even though Alexa 350, 488, 594 and 647 both share the same family name, they each belong to a different chemical family (coumarin/pyridine, rhodamine, cyanine etc).

Like most people, fluorophores too have all their own individual strengths and weaknesses, their own “personalities”. There has yet to be a technology I’ve found that doesn’t need to be understood in those terms in order to best relate to an experiment, trouble shoot difficulties and interpret data. And, just like people, we don’t need perfection, simply that chemists continue to develop the fluors as if developing their personalities to another level of maturity!