Table 1.

Phore requirements

Application	Methodology	Labeling requirements	Example fluorophore	Problems/recommendations
Conformation change	Intensity change	Site-specific cysteine	MDCC (Coumarin)	Requires an environmentally sensitive fluorophore.
	FRET/FLIM	Site-specific cysteine	Cy3/Cy5	Requires sufficient spectral overlap and fluorescent proteins are best for large rearrangements.
		fluorescent protein	GFP/RFP
	Single-molecule FRET	Site-specific cysteine	Cy3/Cy5	Fluorophores must be photostable and bright.
Protein–protein interactions	Intensity change	Site-specific cysteine	MDCC (Coumarin)	Requires an environmentally sensitive fluorophore.
	Anisotropy	Site-specific cysteine	Fluorescein	Label must be small and fluorescence lifetime ∼5 nsec.
		N-terminal amine		Can be attached away from interaction site.
		Peptide tag
	FRET/FLIM	Site-specific cysteine	Fluorescein/rhodamine	Fluorophores can be positioned away from the site of interaction to prevent interference.
		Fluorescent protein	GFP/RFP
	Single-molecule FRET	Site-specific cysteine	Cy3/Cy5
Single-molecule/particle tracking	Single-molecule imaging	Site-specific cysteine	Cy3B	Fluorophores can be spaced away from protein and targeted to inert areas.
		Peptide tag
		N-terminal amine
		Biotinylation	Quantum dot	Large label so may interfere with activity.
				Can be used in live cells with recombinant proteins.
		Fluorescent protein	eGFP	Use only for rapid processes because photobleaching is fast.
				Fluorescent proteins are easiest to use for live cell measurements.
Protein counting	Photobleaching	Fluorescent protein	eGFP	Use monomeric fluorescent proteins. Only fluorescent proteins give a defined stoichiometry.
Live cell localization	Live cell imaging	Fluorescent protein	eGFP	Use monomeric fluorescent protein.
		Peptide tag	FlAsH	Organic fluorophores are brighter and more stable than fluorescent proteins but the labeling is less specific.
		Peptide tag	SNAP-Tag