Fluorophore Selection Guide

With so many fluorophores now available, choosing between them can seem daunting. This article provides a recap of factors to consider for fluorophore selection, before looking at some of the newer fluorophores to reach the market and the problems they are designed to resolve.

Advantages and applications of fluorescent detection

According to Jacqueline Steenhuis, Ph.D., Technical Application Scientist at Biotium, fluorescent detection in life sciences uses two main types of reagents. “Through reactive groups, fluorescent dyes can be used to label proteins such as antibodies or other biomolecules,” she says. “Fluorescent probes instead target specific molecules or organelles based on their physical properties. Fluorescence is very attractive for detection because of its sensitivity, high signal-to-noise, opportunity for multiplexing, and the potential for quantitative detection over a wide linear range. As a result, fluorescence is used for techniques spanning flow cytometry, microscopy, and western blotting, through to ELISA, enzyme activity assays, and qPCR.”

Challenges for fluorophore selection

“Selecting a fluorophore that is appropriate for the sample type, application, and detection platform is often challenging and confusing,” says Taryn Jackson, Product Management Specialist at Thermo Fisher Scientific. “Some fluorescent reagents can be used on cell and/or tissue samples, while others are suitable for solution assays, detecting certain biomolecules such as nucleic acids on gels, or for conjugation to proteins. Often the same fluorophore is available in various forms to target different biological structures, functions, and molecules. Fluorophores for cellular staining also have multiple properties—some can stain live cells, others can stain fixed cells, some stain live cells but are fixable, so it can be difficult to ensure that the fluorophore selected is suitable for the sample preparation method. It’s also important to make sure that the detection platform and its filters are compatible with fluorophores since a fluorophore that is optimal for flow cytometry, imaging, or microplate assays may not work well for all applications. It’s much easier to get the right fluorophore for your current instrument than to update the instrument and/or its filters to match the dye.”

Mike Blundell, Product Manager at Bio-Rad, notes that multiplexing introduces additional problems. “Multi-laser excitation is common for most fluorophores and the resultant emission into other channels will be the cause of spillover in a panel,” he explains. “For example, everyone knows PE is optimally excited by the 561 nm laser, and that you can use the 488 laser if you don’t have the 561 laser, yet not everyone will know that PE is excited a small amount by the 355 and 405 lasers. This means every PE tandem will also have this excitation. Working out how much spillover there is and picking both the right fluorophore and marker can be the difference between a good and a bad panel.”

Key fluorophore properties to consider

When selecting fluorophores, researchers instinctively look at the excitation and emission maxima. However, there are many other fluorophores properties that should be considered. “Photobleaching is a common problem for fluorescent microscopy, so understanding the photostability of your dye is important,” says Danielle Callahan, Director of Product Management at Proteintech. “The size of the fluorophore may also be of significance for steric hindrance and penetration of thick tissues or into the nuclei of cells. In addition, brightness can be critical when assigning fluorophore-labeled antibodies to cellular markers based on target abundance.”

Safa Moreno, Associate Product Manager, Cell Analysis at BioLegend, notes that heat and fixation stability are other key considerations. “Fixation stability is especially pertinent when working with fluorescent proteins, such as PE or APC and their tandems, which may be more susceptible to alcohol-based fixation methods than small molecule or polymer dyes,” she says. Steenhuis adds that membrane permeability, water solubility, toxicity, autofluorescence of biological samples, and compatibility with mounting media can all make the difference to the performance of a fluorophore in an experimental setting.

Factors to consider for fluorophore selection

• Spectral profile—the excitation and emission maxima should be compatible with the instrument’s lasers and detectors, and with other fluorophores in a multiplexed panel; selecting fluorophores with narrow spectral bandwidths can help to reduce spillover
• Brightness—useful when assigning fluorophores to cellular markers based on target abundance
• Stokes shift—the difference between the excitation and emission maxima, which can be exploited when the number of available lasers becomes limiting
• Fluorophore size—smaller fluorophores may be preferred for penetration of thick tissues or into the nuclei of cells
• Photostability—especially critical for fluorescent microscopy, including live-cell imaging applications
• Heat stability—important in long-term and time-lapse live-cell imaging
• Fixation stability—fluorescent proteins may be more susceptible to alcohol-based fixation methods than small molecule or polymer dyes
• Stability over time and from batch-to-batch—tandem dyes can be especially prone to variability
• Specificity—non-specific binding of fluorophores can be due to chemical properties such as charge, hydrophobicity, and fluorophore type; for example, cyanine dyes and their tandems are known to bind non-specifically to monocytes
• Toxicity—fluorophores used in live-cell imaging should have minimal toxicity and not affect cellular health or function
• Autofluorescence of biological samples—fluorophores with red or far-red emissions, or narrow emission spectra, are useful when working with samples that have high autofluorescence
• Membrane permeability—this will determine whether there is a need for fixation and permeabilization if detecting intracellular targets
• Water solubility—high water solubility helps to prevent aggregation or precipitation of fluorophores and fluorophore-labeled reagents out of solution, as well as facilitates a greater degree of labeling for increased brightness
• Staining buffer requirements for multiplexing—some fluorophores require specific buffer conditions to prevent aggregation when combined with other fluorophores
• Compatibility with mounting media—some fluorophores are not compatible with certain antifade reagents
• Ability to pre-mix—useful to reduce pipetting errors between samples (and can save time /money) if building large panels and having samples arrive over an extended period

Newer fluorophores address common problems

Modern fluorophores are designed to overcome known issues for fluorescent detection. “BioLegend’s fluorophores include PerCP/Fire™ 780, which expands blue laser-excited options for multicolor panels and is ideal to be paired with antigens with low expression levels due to its strong brightness,” says Moreno. “We also offer PE/Fire™ 810, APC/Fire™ 810, and PerCP/Fire™ 810, which extend the range of spectral detection further than any previous fluorophores by emitting deep in the far red. Furthermore, BioLegend developed Spark Dyes—simple organic fluorophores with relatively narrow excitation/emission spectra designed to fit in unoccupied spectral spaces. Spark Dyes exhibit limited spillover and, due to their synthetic nature, they are not typically sensitive to standard fixatives, making them useful for a wide variety of applications, including phospho-flow and imaging.”

Blundell highlights Bio-Rad’s StarBright dyes, which are designed to be bright yet also have improved spectral characteristics so that the brightness does not increase spillover and compromise resolution. “StarBright dyes are extremely stable, with narrow excitation and emission spectra and unique spectral signatures, which are consistent in a wide range of experimental procedures including full-spectrum flow cytometry,” he says. “They are also fixable and can be pre-mixed with no special buffer required. Being able to pre-mix helps to reduce pipetting errors between samples, which can be especially useful if building large panels and having samples arrive over an extended time period.”

Biotium’s flagship line of fluorescent CF^® Dyes for labeling antibodies and other proteins is engineered to address several challenges for fluorescent dye bioconjugates. “CF Dyes are highly water-soluble, allowing more dyes to be conjugated to each probe for brighter conjugates,” explains Steenhuis. “In addition, our proprietary dye chemistry has allowed the development of highly photostable CF Dyes with a wide range of emission colors.” The CF Dye range encompasses dyes with optimal photoswitching properties, including several green-excited dyes developed to complement the red-excited dyes used for multicolor dSTORM, and near-IR CF Dyes that improve on early commercial near-IR dyes by exhibiting less non-specific probe binding, improved stability, and better biocompatibility.

“Our CoraLite^® Plus fluorophores are chemically engineered with SuperHydrophilic dPEG^® (discrete polyethylene glycol) groups to increase their solubility and stability,” reports Callahan. “Increased solubility eliminates aggregation or precipitation of the fluorophores or labeled antibodies out of solution, while the dPEG chains also facilitate greater degrees of labeling and stability of the dyes, resulting in increased brightness. We use the same CoraLite Plus dyes for our directly conjugated primary antibodies, our FlexAble Antibody Labeling Kits, and our new Multi-rAb Recombinant Secondary Antibodies (mixtures of recombinant monoclonal antibodies that recognize multiple epitopes on the same IgG) to give researchers as many detection options as possible.”

“Thermo Fisher Scientific’s fluorophore products include our CellEvent Caspase-3/7 Green and Red Detection Reagents, which were developed as fluorogenic no-wash apoptosis indicators to eliminate the risk of losing fragile apoptotic cells,” says Jackson. “These reagents consist of a four-amino acid peptide conjugated to a nucleic acid-binding dye, and are simply added to live cells, where their cleavage by caspase-3 and -7 enzymes activated during early apoptosis results in a measurable fluorescent signal. We’ve also developed pHrodo reagents, a range of fluorogenic dyes that increase their fluorescence as the pH becomes acidic, making them ideal for visualizing acidic compartments in the endocytic or phagocytic pathways, monitoring pH changes in the cytosol, or tracking uptake of antibodies.”