Choosing the right fluorophores for your experiment

Selecting the right fluorophore can be difficult. An experiment requiring only one fluorophore is relatively simple, as we only need to consider the molecule's excitation and emission spectra. However, it may take some optimization to find the best fluorophore for the particular application (Table 1).

Table 1. Labels and their most common applications

Designing a multiplex panel with two or more fluorophores can be more challenging as we must consider the spectral overlap between fluorophores, among other factors, to minimize potential problems such as fluorescence spillover and compensation errors.

Fluorescence spillover is a common issue in which fluorescence that should only be detected in one channel is also detected in an adjacent channel. It is caused by an overlap between the emission spectra of the fluorophores being used and can cause inaccurate results.

Compensation is commonly used to correct for fluorescence spillover, but this can also introduce errors. Selecting the right fluorophores is essential for minimizing fluorescence spillover and ensuring accurate results. Using tandem dyes can also help prevent spillover (see next section).

We have put together a fluorochrome chart to guide you through each step involved in selecting a fluorochrome to make the process quick and easy. The chart features the 30 most popular fluorescent labels, allowing you to quickly select the most suitable fluorophores for your next multiplex experiment.

Our abcam fluorophore chart includes:

• Aligned emission and excitation fluorescence spectra for 30 of the most commonly used fluorophores, including tandem dyes
• Easy visualization of some of the most popular lasers and filters across the fluorescence spectra
• The fluorescence channel and relative brightness for each of the fluorophores
• A short guide with examples

See our Abcam fluorophore chart below, to help you choose the most suitable labels for your multicolor panel.

Tandem fluorescent dyes are conjugated 'dual' fluorescent molecules consisting of two different fluorophores that are covalently attached and close enough for energy to be transferred between them via fluorescence resonance energy transfer (FRET). They are beneficial when designing experiments requiring several different colors. Tandem dyes can also help prevent problems with fluorescence spillover by increasing the separation of the excitation and emission spectra so they do not overlap.

As shown in figure 3, the fluorophore acting as a donor absorbs and is excited by the light energy of a specific wavelength. Upon excitation of the donor, energy is transferred to the acceptor through FRET due to their proximity. The fluorophore acting as an acceptor then emits the transferred energy as fluorescent light at a characteristic longer wavelength.

For example, in phycoerythrin-Cy7 (PE-Cy7), PE and Cy7 act as the donor and acceptor fluorophores, respectively. The laser excites the donor molecule only (PE) – it is not the correct wavelength to excite the acceptor. The donor molecule's emission energy is the right wavelength to excite the acceptor molecule, Cy7, which then releases energy in the form of a photon at its signature wavelength. Therefore, PE-Cy7 has the excitation wavelength of PE fluorophore, but the emission characteristics of Cy7.^.2

Using tandem dyes can increase your panel size and flexibility. This is because when using tandem dyes, a single laser can excite several fluorophores, which are measured by different detectors. For example, Alexa Fluor® 488, PE, PerCP-Cy5.5, and PE-Cy7 are all excitable with a blue laser (488 nm). However, they will produce green, yellow, purple, and infrared emissions (Figure 6).

• Always protect tandem dyes from light as they are highly susceptible to photobleaching
• Do not freeze tandem dye antibody conjugates, as this may result in the denaturation of the donor fluorophore
• Minimize sample fixation or permeabilization as much as possible, as this reduces their brightness
• Each batch requires optimization due to high batch-to-batch variation
• Bleed-through emission from the donor may occur as FRET efficiency is not 100%
• Label cells at 4°C to avoid degradation or decoupling of tandem dyes

Fluorescent proteins (FPs) are naturally occurring proteins with fluorophores incorporated into their structure. They are commonly used in biological imaging and as biosensors in live cells. FPs can be fused to a protein in transgenic cells or animals, conjugated to an antibody, or even used as a substrate in enzymatic reactions. When combined with FRET, FPs can also provide high-resolution information about the proximity of proteins within cells, overcoming the theoretical resolution limit of optical imaging.

Since the original green fluorescent protein (GFP) gene was cloned from the jellyfish Aequorea victoria in 1992³, the variety of fluorescent proteins available for research has increased significantly. In addition to GFP and its variants, other commonly used fluorescent proteins include phycoerythrin (PE), R-phycoerythrin (R-PE), and allophycocyanin (APC), along with naturally occurring and genetically modified fluorescent proteins from a range of species

There are many factors to consider when selecting a fluorescent protein. Below, we have outlined all of the key considerations, discussing why they are important and how they can influence your choice of the fluorescent protein.

Once you understand the properties you need for your specific application, you can choose the best protein using our quick reference card (below), which details some of the properties of the most popular fluorescent proteins.

Fusing the fluorescent protein to the C- or N-terminus of your protein of interest largely depends on the protein itself, including how it folds and whether the terminus has a functional role. For example, if the C-terminus is folded inside the protein, you're unlikely to receive any fluorescence signal. If your protein is post-translationally cleaved close to the terminus your fluorescent protein is fused to, it will be removed from the protein of interest.

For a new experiment, it is ideal – if resources are available – to clone both C- and N-terminal tagged constructs to determine the best option. One research group found that more C-terminal fusion proteins localize to the intended subcellular compartment than N-terminal tagged fusion proteins.⁴ However, it is important to stress that while C-terminal tagged proteins tend to localize and behave as expected, this is not always possible to predict.

You should confirm fusion protein localization by using an antibody against the native protein in the following ways:⁵

• Immunofluorescence to check that the fusion protein localizes correctly
  
• Immunoblotting to confirm the fusion protein is the correct size and expressed at the expected levels
• Co-immunoprecipitation to assess how the fusion protein interacts with known binding partners

If you plan on using multiple fluorescent proteins in the same experiment, choose fluorophores with distinct emission peaks that can all be excited with the lasers available on your equipment. Differentiating the signals can be difficult or even impossible if the emission peaks overlap.

Typically, the brightest FP within your available spectra is best to achieve a clear signal and overcome any background fluorescence. Brightness values are a product of the protein's extinction coefficient, which describes the amount of light it absorbs at a given wavelength, and its photon emission efficiency, known as its quantum yield. However, the resulting number can be difficult to interpret, so an FP's brightness relative to a well-defined fluorescent marker, like EGFP, is a standard alternative measure.

Maturation defines how long it takes an FP to fold correctly, form the fluorophore, and fluoresce. A short maturation time can be important for time-sensitive events in live cells. Superfolder GFP (sfGFP) can fold in under 10 minutes, while mOrange2 can take over four hours to fold correctly.

Bleaching is a measure of photostability, or how long after excitation, the chromophore loses the ability to emit light. The most common measurement of photostability is bleaching half-life (t½), the time taken for an initial emission rate (measured in photons per second) to reduce by half. If you plan on conducting lengthy time-lapse experiments, consider an FP with high photostability. T-sapphire has a bleaching t½ of 25 seconds, but EGFP is much more stable, with a bleaching t½ of 174 seconds.^.6

Like most proteins, FPs are affected by pH, temperature, and oxygen levels. Depending on the environment in which you plan to use your FP, you may need to adjust the conditions slightly or select a more appropriate fluorophore.

The pH can affect excitation and emission peaks, and most FPs are sensitive to acidic conditions. Some FPs can change fluorescent intensity upon pH changes, such as pHTomato, which may be experimentally useful. The pKa value is a good indicator of pH sensitivity, showing the pH at which half of the chromophores are fluorescent.

Temperature and oxygen levels both affect maturation times. Hypoxic conditions tend to delay maturation times, as do temperatures outside the FP's optimal range. However, newer FPs like UnaG, a green fluorescent protein isolated from the Japanese freshwater eel (Anguilla japonica), fluoresce even when oxygen levels are low.⁷

Most FPs are derived from jellyfish or coral proteins rather than the species in which they're most likely to be used experimentally. This can result in poor FP expression and low fluorescence signal due to interspecies differences in amino acid codons usage.

Fortunately, many new versions of FPs have been codon-optimized to reflect mammalian cell preferences. For example, Jürgen Haas and colleagues improved the signal from GFP up to 120-fold by modifying the codon sequence.⁸

If you're using an older plasmid to generate your fusion proteins, it may not contain a mammalian-optimized FP sequence. Check whether your FP sequence has been modified for use in your experimental species.

It's essential to determine whether your fluorescent protein is a monomer or dimer and whether this affects your experiment. Many of the early FPs were prone to forming oligomers which can affect the biological function of the fusion protein. EGFP, for example, is a monomer that can form dimers when used in high enough concentrations, potentially distorting subcellular organelles or disrupting experiments like FRET.^9,10 Monomers are usually denoted by an 'm' prefixing the protein name, such as mCherry, and truly monomeric FPs are recommended in most cases.

These considerations are summarized in table 1 below.

Table 1. Factors to consider when selecting a fluorescent protein