Considerations for Selecting a Fluorescent Dye or Ligand

Throughout their history, fluorescent dyes have enabled the visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists. Seeing inside these specimens has offered illumination on biochemical processes that are crucial in the world of biological research, medical therapy, and even drug development (Grimm & Lavis, 2022). With the wide breadth of fluorescent tools that have been developed throughout the past century, it can be overwhelming to select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting to the most popular or familiar options without even realizing it. Yet, this selection is crucial as it will impact downstream outcomes of an experiment, including the accuracy and efficacy of findings. If you’re struggling to select the right fluorescent tools, this blog is here to help. We will outline some fundamental characteristics to consider when selecting a dye or ligand to make your selection easier, as well as highlight some recent advancements within this field. 

Figure 1: Example structures of fluorophores, fluorescent dyes, and fluorescent ligands. Figure created with biorender.com.

Fluorescence refers to the emission of light by a substance (fluorophore) that has absorbed light or electromagnetic radiation at another wavelength (Heinrichs, 2009)). When a fluorophore absorbs light, electrons are elevated from a ground state to an excited state through the absorption of photons. After a brief elevation in this excited state the electron returns to its ground state by emitting a photon with slightly less energy due to non-radiative energy losses. This difference in emission results in a slightly longer wavelength emitted than was absorbed. This phenomenon is known as the Stokes Shift, or the difference in wavelength between peak excitation spectrum and emission spectrum. Fluorescent dyes utilize these fluorophores by combining them with a molecular species with high affinity for an endogenous molecular target, meaning the dye will be attached to the target through binding of the molecular species. Administration of these dyes results in direct binding between the dye and the endogenous target, and thus no genetically encoded tag is required. These dyes can also be attached to an antibody to detect various targets, such as proteins, nucleic acids or glycans.

Fluorophores can also be covalently attached to self-labeling proteins. Some common examples of these self-labeling proteins would be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme variant tag that covalently reacts with a small substrate ligand motif attached to a fluorophore. When paired with a protein of interest, these tools provide an easier means to identify targets through fluorescent labeling. There are several advantages to using these self-labeling proteins over traditional fluorescent protein tags, such as higher specificity, increased versatility in probe attachment, and compatibility with far-red and near-infrared fluorophores. 

Fluorescence microscopy takes advantage of the characteristics of these fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent dyes and ligands vary in their chemical structure, leading to differences in their excitation and emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These variations directly impact the way one can utilize these dyes. The most used fluorescent dye is fluorescein isothiocyanate (FITC), with excitation/emission peak at 495/517 nm. FITC binds to an amine on a protein, enabling the labeling of protein substrates/peptides and antibodies. Example applications of this due include coupling to an antibody, whereby excitation of blue light will indicate where FITC is bound through green light emission. 

• Effects on Protein Function and Cellular Components: It's important to assess how dye-conjugation affects protein function and its propensity to bind cellular components. Some dyes target specific organelles like mitochondria, and different dyes can result in varying cytoplasmic background signals when used with the same protein (Hayashi-Takanaka et al., 2014). Other dyes may prove challenging to stain intracellular compartments in in vivo model systems, so extra care must be taken to assess proper staining is achieved. Fluorescent ligands have more flexibility due to the affinity tag they typically bind to.
• Stability of Fluorescence: For applications like that involve ranges of pH, and significant lengths of time, the stability of fluorescence over time is crucial. For instance, the fluorescence of fluorescein isothiocyanate (FITC) can be stably combined with nanoparticles, maintaining intensity at ~85% of its original value after 60 minutes of illumination, highlighting its reliability for long-term experiments (Salari et al., 2019). Photostability, the ability to retain fluorescent intensity during exposure to light, is another factor to consider. If your application requires longer periods of exposure, you’ll want a more photostable fluorescent protein (Mahmoudian et al., n.d.). Factors such as exciting the fluorophore at peak wavelength can also decrease the rate of photobleaching.
• Interaction with Receptors: Understanding how fluorescent dyes interact with specific receptors is key, especially for G protein-coupled receptors (GPCRs). Mutations in receptor sites can affect the fluorescence emission of labeled agonists and antagonists, impacting the interpretation of experimental results. It's important to consider how dyes can influence receptor conformation and signaling activity (Sridharan et al., 2014). It's also crucial to evaluate how the addition of fluorophores affects the functional activity of the ligands. For instance, studies have shown that different fluorescent ligands can elicit varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency and efficacy of these ligands can be affected by the incorporation of fluorophores, as seen in studies involving dopamine D2/D3 receptors (Allikalt et al., 2020).
• Impact of Labeling Process: The process of dye labeling, such as conjugation with proteins, should be carefully managed. Proper dye-to-protein ratios, or the degree of labeling (DOL), and thorough removal of unconjugated dye molecules are critical for accurate and reliable imaging and assay results (Hayashi-Takanaka et al., 2014). Conjugates with lower DOL tend to have weaker fluorescence intensities, and the best practice to determine optimal DOL is through several small-batch experimental labeling. Size is also a factor. Bulkier tags, such as GFP, may interfere with protein expression or function in live cells.
• Fluorophore Properties: The specific properties of the fluorophore, such as excitation wavelengths and emission profiles, need to be matched with the experimental setup and objectives. Most systems can only excite and read a set number of excitation and emission wavelengths, as this is limited by the filter cubes and filtering settings of the equipment. Systematic evaluation of commercially available dyes with different excitation wavelengths for green, red, and far-red dyes is important for intracellular fluorescence live imaging (Hayashi-Takanaka et al., 2014). Dyes can also vary in their brightness, depending on the respective fluorochrome. Brightness is impacted by both the excitation coefficient, characterizing the strength a species absorbs or reflects light or radiation at a specific wavelength, and the fluorescence quantum yield, the ratio of photons absorbed over the photons emitted. For example, DAPI is much dimmer than Alexa Fluor 488, and thus it is more difficult to visualize the stain from the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. 
• Experimental Context and Ligand Binding: The context of the experiment, such as the presence of cells or specific proteins, can influence the choice of fluorescent dye. For instance, the cleavage of FITC-conjugated protein ligand is unaffected by additional protein from cells under certain conditions, which is an important consideration for assay design (Breen et al., 2016). When selecting dyes, it's important to test them under experimental conditions. Common issues include photobleaching, non-specific binding, and quenching, which can be mitigated through careful selection and optimization.
• Fluorescent Experiment Types: Different types of fluorescent experiments, such as using fluorescently tagged ligands or intermolecular FRET for ligand-receptor interactions, require specific considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend specific fluorescent donors and acceptors in their product descriptions, or technical manuals. 

For effective live cell imaging, the use of dyes that exhibit low toxicity and high photostability is crucial. These properties ensure a more consistent and stable signal over extended periods, which is particularly beneficial when studying dynamic processes like mitochondrial dynamics. This process, involving mitochondrial homeostasis maintained through mechanisms like fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like Rhodamine 123 (R123) (Liu et al., 2017). In multi-color imaging, it is essential to select dyes with different emission spectra to avoid crosstalk/crossover to reduce ‘muddying’ of the signal and facilitate clear differentiation between multiple dyes during analysis. 

Image resolution is impacted by several factors. Firstly, brighter dyes enable detection of finer details by improving signal-to-noise ratios. Certain dyes have been specifically designed for use in super-resolution microscopy techniques, such as Stimulated Emission Depletion (STED), and Stochastic Optical Reconstruction Microscopy (STORM). These dyes have specific properties that enable resolution beyond the diffraction limit of light. Dyes like silicon-rhodamine or Janelia Fluor (JF) dyes have specific properties that position themselves for super resolution usages, such as superior brightness, photostability, and most importantly, a photoswitching ability. This photoswitching refers to the ability to ‘toggle’ between fluorescent and non-fluorescent states in response to a specific stimulus, which leaves only a small volume of fluorophores in an excited state, effectively reducing the area of fluorescence. 

When it comes to protein labeling and analysis, dyes should be small enough not to hinder protein function and should possess high specificity to ensure accurate tagging. In Fluorescence Resonance Energy Transfer (FRET) applications, the dyes need to have compatible excitation and emission profiles to enable effective energy transfer, which is key to observing interactions and conformational changes in proteins. Both short distances of molecules (<10nm) and overlap of excitation profiles is vital for the accurate interpretation of FRET-based experiments. A popular combination is Alexa594, and Alexa647 due to their brightness and high degree of overlap.

Dyes for DNA/RNA staining should have high affinity and specificity to nucleic acids, with minimal background staining. Additionally, cell permeability is a critical factor, as it influences the dye's ability to enter cells without causing undue toxicity. A prominent example is DAPI, a dye that binds specifically to the AT-rich regions of double-stranded DNA and exhibits a blue-fluorescent light when excited. While it is technically cell-permeable, its use in live cells can sometimes result in cellular toxicity due to the permeation process.

Figure 2: Excitation and emission spectra and table for the HaloTag® Janelia Fluor Ligands.

Throughout the history of cell biology, fluorescent labeling techniques have significantly enhanced our understanding of cellular mechanisms. Advancements in dye chemistry have led to the development of more stable, brighter, and specific dyes, expanding the capabilities of fluorescence-based techniques in biotechnology. Recent examples of advancements include the Janelia Fluor® (JF) dyes developed by the Lavis Lab (Grimm et al., 2017). These dyes were adapted into ligands for use with the HaloTag® technology and are currently offered at Promega. Due to the versatility with interchangeable labeling using the HaloTag® system, the new Janelia Fluor® HaloTag® Ligands expand the self-labeling capabilities of this platform. The Janelia Fluor® ligands provide brighter and more stable fluorescence for super-resolution applications. Other current advancements include work exploring far-red-emitting dyes in bacterial imaging, offering solutions to overcome the limitations posed by autofluorescence (Lucidi et al., 2023).

One specific application in dire need of advancements in fluorescent technology is the detection of endogenous proteins. Fluorescence intensity of traditional dyes or ligands, such as mCherry, can lead to challenges when attempting to detect very low levels of endogenous protein. Below we show an experiment where mScarlet and HaloTag (+JF549) were knocked into the HIF1α (hypoxia inducible factor 1 subunit alpha) locus to compare fluorescence expression on a low-level endogenous protein. These clones were treated with 1µM of the proteasome inhibitor, MG132, for 6 hours and then imaged (Figure 3). A comparison of the two clones clearly demonstrates a brighter fluorescence in the HIFα-HaloTag (+JF549) clones when compared to the mScarlet clone. In a separate experiment, a low stimulating concentration of Phenanthroline (2µM) was administered and fluorescence was imaged after 6 hours. Even under low concentration of Phenanthroline, there was much greater fluorescence levels in the JF549 clones when compared to the mScarlet clones. One of the most significant advantages of the Janelia Fluor® dyes is their exceptionally higher fluorescence brightness compared to conventional dyes.

Figure 3: JF549 is more sensitive than mScarlet; thus, enabling the detection of endogenous HIF1α accumulated under normal condition or at low concentration of phenanthroline. The top row is under normal condition (+1 µM MG132). The bottom row is under stimulation of phenanthroline (+2 µM).

The field of fluorescent ligands and dyes has witnessed remarkable advancements, driven by the need for more precise, stable, and versatile imaging tools in biological research. This does make finding the right dye for your experiment more complicated. We hope this post helps you in your search for the perfect way to light up your science. 

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