What fluorophores should I use for my experiment?

Oh, this is a great question - and answer: it really depends on what you want to image. For example, I am looking at trafficking of proteins into the cell wall. The cell wall usually has a lower pH, often around 4.5 -5.5, so I would be looking at fluorophores that have a pKa around that range; e.g. mCherry (pKa 4.5), mRuby3 (4.8), GreenLantern (pKa 5.6), mNeonGreen (pKa 5.7) or Citrine (pKa 5.7) would be great candidates. I also need a fluorophore that folds fast so the fluorophore is functional in the endomembrane system and I can visualize the protein trafficking. mCherry, GreenLantern and mNeonGreen all are predicted to fold within 15 mins, at least at regular cell culture conditions around 37C, while mRuby3 is a slow folder, with more than 2 hours until maturation; so the latter is not as good a choice for this experiment.

The choice of fluorophore for your experiment depends on the microscope you will use, the tissue/cell line/cellular compartment of interest, and the temporal scale of the experiment. Fluorophores are continuously updated by the community, and besides their range of excitation and emission wavelengths, they vary in several important parameters, such as folding time, brightness, lifespan, tendencies to form multimers, and pH sensitivity/pKa, amongst others. These parameters might also vary depending e.g. on temperature or mounting media used for the experiments. For an extended list, please go to: https://www.fpbase.org/.