What to expect

• Aligned emission and excitation fluorescence spectra for 30 of the most commonly used fluorochromes, 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 fluorochrome
• A short guide with examples

An introduction to fluorochromes: Excitation and emission spectra

Fluorescent dyes (or fluorochromes) are commonly used as detection reagents in various applications such as cellular imaging and flow cytometry. Fluorochromes can include advanced types such as Alexa Fluor dyes, quantum dots, and fluorescent proteins, as well as molecular probes designed for specific detection. Fluorochromes absorb light energy of a specific wavelength (excitation wavelength) and re-emit it at a longer wavelength. The process involves absorbed photons exciting electrons from the ground state to the excited state; as electrons return to the ground state, the remaining energy is released as emitted photons with less energy, resulting in longer wavelengths (the emission maximum) due to some energy being lost as heat. The wavelengths at which the fluorochrome absorbs and emits light are known as the fluorochrome’s excitation spectrum (range of excitation wavelengths) and emission spectrum (range and peak, or emission maxima/emission maximum, of emitted light), respectively (or just fluorescence spectra).

Instruments requiring fluorescent dyes, such as fluorescence microscopes and flow cytometers, are equipped with lasers producing light at a particular wavelength (often at shorter wavelengths) so they can excite fluorochromes capable of absorbing light at that specific wavelength. UV light is sometimes used to excite certain fluorochromes, and different fluorochromes can be excited by other wavelengths. The light emitted by the fluorochrome is then filtered using optical, emission, and emission filter sets so that each sensor will detect fluorescence only within the filter’s range. The selection of the correct filter sets is crucial for accurate detection and to prevent bleed-through in fluorescence microscopy and flow cytometry. This fluorescence is the read-out signal provided by the instrument.

Many different fluorochromes have been developed, each one with a particular emission and excitation fluorescence spectrum. Some fluorochromes are designed to bind to free molecules such as DNA, and each given fluorochrome has unique fluorescent characteristics, including its emission and excitation properties, which are important for experimental design. You can find commercially available fluorescent dyes that absorb and emit light at specific wavelengths across the whole visible spectrum, including the infrared region. The emission and excitation properties of fluorochromes can be visualized as excitation and emission spectra, and emission maxima are critical for selecting the right dye for a given application. Fluorochromes differ in the intensity at which they emit light. Therefore, the fluorochrome brightness will depend on its ability to absorb light and the efficiency with which the absorbed light is converted into emitted light.

Choosing the right fluorochromes for a mutliplex panel

When designing a multiplex panel for flow cytometry experiments, carefully selecting fluorochromes is essential to ensure accurate and reliable results. Each fluorochrome has distinct excitation and emission spectra, determining the light wavelengths it can absorb and emit. In multiplex panels, where multiple fluorescent molecules are used simultaneously, choosing fluorochromes with minimal spectral overlap in their emission spectra is crucial. Overlapping emission wavelengths can lead to fluorescence spillover, where the emitted light from one fluorochrome is detected in the channel intended for another, potentially compromising data quality.

To address these challenges, researchers rely on comprehensive fluorochrome charts that display the excitation and emission properties of different fluorochromes. These charts help visualize the emission and excitation spectra, making selecting combinations with minimal overlap and optimal separation easier. Additionally, considering each fluorochrome's relative brightness and excitation properties ensures that even rare events or low-abundance markers can be detected with high sensitivity. By carefully evaluating the emission and excitation characteristics, as well as the relative brightness of each dye, researchers can design multiplex panels that maximize the detection of multiple targets while minimizing interference from fluorescence spillover. This strategic approach is key to successful flow cytometry and the accurate analysis of complex biological samples.

Tandem dyes: Expanding multiplexing capabilities

Tandem dyes have revolutionized flow cytometry by enabling the simultaneous detection of a greater number of cellular markers within a single experiment. These specialized fluorescent molecules are composed of two linked fluorochromes: a donor and an acceptor. When the donor fluorochrome absorbs excitation light, it transfers the absorbed energy to the acceptor fluorochrome through a process known as Förster resonance energy transfer (FRET). The acceptor then emits light at a longer wavelength, resulting in a large Stokes shift and a bright fluorescent signal.

This unique mechanism allows tandem dyes, such as the popular Brilliant Violet dyes, to be typically excited by a single laser, often the violet laser, while emitting light at wavelengths distinct from the donor. This property significantly expands the range of detectable parameters in flow cytometry experiments, as more than one fluorochrome can be excited and detected without the need for additional lasers. Tandem dyes are especially valuable in complex biological research, where the ability to analyze multiple critical markers simultaneously is essential.

However, the use of tandem dyes requires careful planning. Their excitation and emission properties can be influenced by biological conditions and the presence of other fluorochromes, and their emission spectra may have considerable overlap with other dyes. Understanding the specific excitation and emission characteristics of each tandem dye, as well as their quantum yield and relative brightness, is crucial for optimal panel design. By leveraging the advantages of tandem dyes and integrating them thoughtfully with other fluorochromes, researchers can achieve high sensitivity and robust data in advanced flow cytometry applications.