How do you choose which is the best fluorophore combination?

Choosing the Best Fluorophore Combination

To select the best fluorophore combination for a given application, such as in microscopy, flow cytometry, or other fluorescence-based techniques, several factors need to be considered:

Spectral Overlap

Excitation and Emission Spectra: It's important to choose fluorophores with well-separated excitation and emission spectra to minimize spectral overlap. This will reduce the chances of fluorescence bleed-through where the emission spectrum of one fluorophore spills over into the detection channel of another.

Fluorophore Brightness

Brightness: The brightness of a fluorophore is determined by its absorption coefficient and quantum yield. A higher brightness can improve detection of low-abundance targets.

Photostability

Photostability: The resistance of a fluorophore to photobleaching (fading) upon exposure to light. More photostable fluorophores are preferred, especially for applications requiring prolonged illumination.

Biological Factors

Biological Compatibility: Fluorophore selection should also take into account the biological system being studied, including pH and the presence of other fluorescent or quenching substances.

Laser and Filter Compatibility

Instrumentation Compatibility: One must ensure that the chosen fluorophores can be excited efficiently by the light sources available and that appropriate filters are in place for the detection of their emitted light.

Availability of Conjugated Antibodies

Conjugation Factors: It's convenient to select fluorophores that are already conjugated to the antibodies of interest or can be easily labeled.

Economic Considerations

Cost: The price of fluorophores and the necessary reagents may also be a factor when choosing the right combination.

Additional Tips

• Use a fluorescence spectral viewer tool to visualize the excitation and emission spectra overlap between different fluorophores.
• Perform a compensation test to correct for spectral spillover if using flow cytometry.
• Prioritize photostability for live-cell imaging where long-term tracking of fluorescence is required.
• Consider the use of tandem dyes where a donor fluorophore is covalently linked to an acceptor fluorophore for an extended range of emission spectra.