Table 1.

Criteria for choosing FPs and reporters.

Property	Design preference	Complications
Spectra	Narrow, and distinct, for separability by filters (see Figure 2)	Spectra (excitation or emission) that are particularly broad, have multiple distant peaks, or overlap greatly (for two FPs) can lead to bleed-through noise
Quantum yield (QY)	High, for brightness	When low, FP is inefficient and dim, requiring more intense excitation
Extinction coefficient	High, for brightness	When low, FP absorbs light poorly, requiring more intense excitation
Maturation time	Low, for expression sensors	When high, protein is slow to develop its chromophore, and not suitable for reporting expression levels dynamically
Aggregation	Low, for image quality	When high, imagery is corrupted by puncta and activity may be altered by dimer/multimer formation
Photobleaching time	Long (slow bleaching), for dynamics	When short (rapid bleaching), dynamic measurements may be corrupted
pKa	Low, for selectivity	When near physiological pH (6–7.4), fluorescence can be sensitive to pH changes, esp. in lysosomes