Small molecule fluorophores can be attached to biomolecules such as proteins for visualization, tracking, and quantitation in a variety of experiments.

There are many small molecule fluorophores out there, such as CF^® Dyes and Alexa Fluor^TM that can power your experiments, so which one should you choose? For some basic experiments the choice of fluorophore is probably not that critical. However, there are other experiments where the properties of different fluorophores will have a huge impact on the quality of your experiment’s results.

CF^® fluorophores were engineered for improved water solubility, brightness, and photostability. These improved properties can make a substantial difference on the data quality generated from experiments such as cell sorting (FACS) and advanced imaging techniques.

 

In this article:

What is a flurophore?

Labeling antibodies with fluorophores

Advances in CF^® fluorophores

Fluorophore brightness

Fluorophore solubility

Fluorophore photostability

Tracking multiple fluorophores

References

 

What is a fluorophore?

Fluorophores are molecules that absorb light of a certain wavelength, which provides energy to reach a higher-energy and unstable excited state. During the fluorophore’s short-lived excited state, it will usually lose a little bit of energy. Then, it will emit light at a shorter wavelength as it returns back down to its ground state (Figure 1).

Figure 1. A fluorophore is excited when it absorbs light, after relaxation it will emit light as it returns to the ground state (right). Typical excitation and emission spectra for a fluorophore, where the peaks indicate the wavelength of light with the highest excitation and emission for that fluorophore, respectively.

 

There are proteins such as GFP (Green Fluorescent Protein) from bioluminescent jellyfish that are naturally fluorophores. However, in this article we’ll be discussing small organic synthesized fluorophores.

Let’s briefly cover some background on fluorophore nomenclature (Figure 2). Fluorophores usually start with some kind of brand name or name that they’re commonly known by. The focus of this article is CF^® dyes, which stands for “Cyanine-based Fluorescence dyes.” This refers to the cyanine core of the molecule around which tweaks are made to give each CF^® dye its unique excitation and emission properties (Torres, 2020).

Fluorophores often end in a number such as “488”, “514”, or “750”, for example. This number refers to the wavelength of light, in nanometers, that excites the fluorophore (Figure 1).

CF^® dyes sometimes end in a letter that provides additional information about the fluorophore. For example, there are 3 CF^®405 dyes: CF^®405S, CF^®405M, and CF^®405L. All three dyes are excited with 405 nm light, but they have different emission wavelengths with “S”, “M”, and “L” referring to the shortest, medium, and longest emission wavelength. “R” in fluorophores such as CF^®660R refers to the rhodamine core of these dyes.

Figure 2. The first part of a fluorophore’s name is usually the brand or common name. Then, there is a number that refers to the excitation wavelength. Lastly, CF^® fluorophores sometimes have an additional letter at the end that provides extra information about that dye.

 

Labeling antibodies with fluorophores

Since this article is focused on CF^® dyes, it is worth pointing out that there is a very quick and reliable method for labeling antibodies using Mix-n-Stain^TM Labeling Kits. We cover that labeling procedure in depth in this article.

Keep in mind that Mix-n-Stain^TM Kits are not the only method for labeling proteins. We cover another common method in this article.  

 

Advances in CF^® fluorophores

There are many different fluorophores out there that you can use in your experiments. This was even true before scientists started developing new CF^® fluorophores. So why did they feel the need to create more dyes?

CF^® fluorophores were engineered for enhanced water solubility, brightness, and photostability. Based on these parameters, CF^® dyes often outperform other commercially-available fluorophores (Biotium, n.d.) (Figure 3 and Table 1).

Figure 3. CF^® fluorophores that are brighter (left) or more photostable (right) than other commercially available dyes.

 

Table 1. CF^® fluorophores that are brighter or more photostable (Biotium, n.d.)

CF® fluorophore	Enhanced property	Comparison fluorophore
CF®405S	Brighter	Alexa FluorTM 405
CF®405M	More photostable	Pacific BlueTM
CF®430	More photostable	Pacific GreenTM, BD HorizonTM V500
CF®440	More photostable	Pacific GreenTM, BD HorizonTM V500
CF®488A	More photostable	FITC
CF®532	Brighter	Alexa FluorTM 532
CF®543	Brighter	Alexa FluorTM 543
CF®568	Brighter, more photostable	Alexa FluorTM 568
CF®570	Brighter	Alexa FluorTM 568
CF®583	Brighter	Cy3.5TM
CF®583R	Brighter	Cy3.5TM, Texas RedTM
CF®594	Brighter	Alexa FluorTM 594
CF®633	More photostable	Alexa FluorTM 647
CF®640R	More photostable	Alexa FluorTM 647
CF®660C	Brighter	Alexa FluorTM 660
CF®660R	More photostable	Alexa FluorTM 660
CF®680R	More photostable	Alexa FluorTM 680
CF®700	Brighter	Alexa FluorTM 700
CF®750	Brighter	Alexa FluorTM 750

 

There are a lot of different CF^® fluorophores; more than 40 cover the visible and near IR spectrum (Figure 4). The availability of so many CF^® fluorophores enable multiplexing experiments where many different fluorophores are used to label different proteins in the same experiment. We’ll give an extreme example of multiplexed labeling towards the end of the article.

Figure 4. CF^® fluorophores arranged according to excitation wavelength.

 

So, with those advantages in mind, let’s cover some actual research examples where the enhanced properties of CF^® fluorophores improved the quality of experiments. 

 

Fluorophore brightness

One experiment that uses labeled antibodies is fluorescence activated cell sorting (FACS), which is also known as flow cytometry. FACS separates a population of cells based on how highly they express different proteins, or markers, on their cell surface. The level of a given protein marker on the cell surface is analyzed by binding to fluorophore-labeled antibodies that recognize the given marker (Figure 5).

Figure 5. Cells are sorted based on fluorescence intensity (top). Those with low expression of a protein marker (blue) will have low signal from fluorophore-labeled antibodies (purple and green) that bind to that marker (left). Cells with high expression of the marker will have higher signal from fluorophore-labeled antibodies (right).

 

FACS is used to separate different types of immune cells. One research group compared many different CF^® dyes on an antibody that recognizes the protein CD4. This group had previously used Alexa Fluor 532 and Alexa Fluor 700 to label CD4 cells. However, when comparing head-to-head with a suite of CF^® dyes they found that 18 out of the 25 CF^® fluorophores outperformed Alexa Fluor 532, and 10 of the 25 CF^® fluorophores outperformed Alexa Fluor 700. So, they improved their FACS method by substituting two of the CF^® fluorophores for Alexa Fluor 532 and 700 (Jiang et al, 2021).

 

Fluorophore solubility

Enhanced fluorophore solubility was intended to improve the efficiency of labeling proteins with dyes. However, fluorophore solubility can also impact how fluorophore-labeled proteins perform in imaging experiments.

One study, from researchers in Japan, compared how different fluorophores impacted the live-cell imaging of antigen-binding fragments (Fab) that interact with a particular histone modification. When the Fabs were labeled with Alexa Fluor 647, the researchers frequently observed that the Fabs were aggregating in cells. In contrast, when the same Fabs were labeled with CF^®640 almost no Fab aggregates were observed (Hayashi-Takanaka et al, 2014).

These results demonstrate how fluorophore solubility isn’t just important during the labeling step, but also for the performance of the labeled protein.

 

Fluorophore photostability

The photostability of fluorophores is really critical for fluorescence microscopy. Essentially, the more times that you image a fluorophore, the more they lose their fluorescence which is known as photobleaching. However, different fluorophores photobleach at different rates. Some stop fluorescing almost immediately whereas more photostable dyes will still work after minutes or even hours of imaging.

Super-resolution microscopy methods tend to be pretty long, so it is really important to pick photostable fluorophores when performing one of these techniques. In short, the resolution of conventional fluorescent microscopy is approximately 200 to 300 nm, meaning that you only see blurry images or totally miss objects that are smaller than that (Valli et al, 2021).

There are many super-resolution methods, including STORM, STED, SIM, 2-Photon, TIRF, and many more. In general, these experiments work by only labeling a subset of the molecules that you’re tracking, then imaging over and over again to see where the labeled protein moves with time. Imaging your sample over and over again is what causes photobleaching, but is also what enables the super resolution. And, that’s why it is critical to use photostable fluorophores in super-resolution imaging experiments.

As you saw in Figure 2, there are a number of CF^® dyes with enhanced photostability, so it is no surprise that these fluorophores perform well in super-resolution microscopy experiments. See Table 2 for a complete list of CF^® fluorophores that have been experimentally validated in super-resolution applications (Biotium, n.d.).

 

Table 2. CF fluorophores validated in super-resolution experiments.

Fluorophore	Validated super-resolution techniques
CF®405S	SIM, TIRF
CF®405M	SIM, 2-Photon
CF®440	STED
CF®488A	STORM, STED, SIM, 2-Photon, TIRF, DNA-PAINT
CF®535ST	STORM
CF®550R	STED
CF®555	STORM, STED, SIM
CF®568	STORM, STED, SIM, TIRF
CF®583	STED
CF®583R	STORM
CF®594	STED, SIM
CF®597R	STORM
CF®633	TIRF, FIONA, gSHRImp, SMT
CF®640R	STED, SIM, 2-Photon, TIRF, FLImP
CF®647	STORM
CF®660R	SMLM, DNA-PAINT
CF®660C	STORM, MINFLUX
CF®680	STORM, SMLM, MINFLUX
CF®680R	STORM, STED, 2-Photon
CF®750	STORM

 

Multiplexing: Tracking multiple fluorophores

In microscopy applications researchers often want to monitor more than a single protein or molecule at a time. To do so, they put different colored fluorophores on different proteins to track them at the same time.

Figure 6. Nuclear protein labeled with red fluorophore and cytoplasmic protein labeled with blue fluorophore.

 

This approach can answer questions such as:

·         Are these two proteins found in the same subcellular compartment?

·         How much of each protein is found in a cell or in a given subcellular compartment?

·         When one protein is eliminated from the cell, what happens to this other protein?

With common microscope setups researchers have long been able to track about two to four proteins at the same time. For example, you could monitor proteins labeled with blue, green, red, and purple fluorophores, respectively, to ask questions such as those outlined above (Figure 7).

Figure 7. Different antibodies with different fluorophore labels (purple, blue, green, and red) are used to determine the location of different proteins (orange circles, squares, oval, and hexagons).

 

However, recent innovations have taken this fluorophore “multiplexing” to another level. PICASSO (Process of ultra-multiplexed Imaging of biomoleCules viA the unmixing of the Signals of Spectrally Overlapping fluorophores) stretches the boundaries of both fluorescent microscopy and scientific acronyms (Seo et al, 2022).

Essentially, these researchers used math to deconvolute signals coming from overlapping, yet unidentical fluorescent spectra. When I said that two to four fluorophores were traditionally used, this was limited by how many fluorophores exist with functionally non-overlapping spectra. This group gets around that limitation with the power of math!

So now, they were able to monitor up to 15 fluorophore-labeled proteins in a given sample. An important experimental detail is that you need to take as many images as you have different fluorophores, so in this case 15 images per sample. Again, this is where having photostable fluorophores is really important so that you’re getting similar fluorescent intensity on image 15 as on image 1.

The photostability and sheer number of CF^® fluorophores made them a popular choice for PICASSO as 7 out of the 15 dyes they used were CF^®. As a proof of concept, they used PICASSO to image 15 different proteins at a time in a specific brain region called the dentate gyrus (Seo et al, 2022). However, this method should be broadly applicable for labeling proteins across many different cell types.   

 

If you’re ready to start using fluorophore-labeled antibodies for your research then check out our decision tree to help guide which dye will work well for your experiments based on its emission color and photostability (Figure 8). Additionally, check out the link below to find Mix-n-Stain^TM Labeling Kits for any of the CF^® fluorophores that we’ve discussed in this article.

Figure 8. CF® fluorophores organized by their emission properties and if they’ve been validated for super-resolution imaging.

 

 

References

Biotium. (n.d.). CF® dyes: Next generation fluorescent dyes for biological research. Retrieved March 16, 2026, from https://biotium.com/technology/cf-dyes/

Biotium. (n.d.). Super-resolution microscopy. Retrieved March 23, 2026, from https://biotium.com/technology/immunofluorescence-microscopy/super-resolution-microscopy/#cfdyeproducts

Hayashi-Takanaka, Y., Stasevich, T. J., Kurumizaka, H., Nozaki, N., & Kimura, H. (2014). Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging. PloS one, 9(9), e106271. https://doi.org/10.1371/journal.pone.0106271

Jiang, J., Li, X., Mao, F., Wu, X., & Chen, Y. (2021). Small molecular fluorescence dyes for immuno cell analysis. Analytical biochemistry, 614, 114063. https://doi.org/10.1016/j.ab.2020.114063

ONI. (2024). Popular fluorophores for dSTORM imaging (Imaging guide, v6). ONI. https://oni.bio/wp-content/uploads/2024/11/Popular-fluorophores-for-dSTORM-imaging_v6_2024-3.pdf

Park, L.M., Lannigan, J. and Jaimes, M.C. (2020), OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. Cytometry, 97: 1044-1051. https://doi.org/10.1002/cyto.a.24213

Seo, J., Sim, Y., Kim, J., Kim, H., Cho, I., Nam, H., Yoon, Y. G., & Chang, J. B. (2022). PICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements. Nature communications, 13(1), 2475. https://doi.org/10.1038/s41467-022-30168-z

Torres, E. (2020, March 3). CF® dyes. What started it all? Part 2. The chemistry of fluorescence. Biotium. https://biotium.com/blog/cf-dyes-what-started-it-all-part-2-the-chemistry-of-fluorescence/

Valli, J., Garcia-Burgos, A., Rooney, L. M., Vale de Melo E Oliveira, B., Duncan, R. R., & Rickman, C. (2021). Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique. The Journal of biological chemistry, 297(1), 100791. https://doi.org/10.1016/j.jbc.2021.100791

 

 

 

Tags

antibodies antibody antibody labeling CF Dyes Mix-n-Stain

• Share :
• Deel
• X
• LinkedIn

---