Table 3. Advantages and Limitations of Labeling Strategies.

Labeling consideration					Fluorophore consideration
Engineering type			Function perturbation	Specificity	Fluorophore	Brightness/photostability	Pros	Cons
Genetic	Genetic labeling		perturb	high	FPs	Lower/lower	Directly labeled on target	Require correct protein folding and maturation
	SNAP/Halo tags		perturb	high but may suffer incomplete dye conjugation	Synthetic dyes	Higher/higher	Directly labeled on target; great flexibility in dye selection	May encounter false signals from trace dye, need extensive cleaning
	NAA		less perturbation	high but may suffer incomplete dye conjugation	Synthetic dyes	Higher/higher	Directly labeled on target	Limited dye availability and SMLM compatibility
Nongenetic	Immuno-labeling	Direct	No	Varies, determined by the antibody used	Synthetic dyes	Higher/higher	Can detect endogenous targets	Limited qualified specific antibodies; potential nonspecific binding and cross-reactivity; not compatible for live imaging
		Indirect	No	Varies, determined by the antibody used	Synthetic dyes	Higher/higher	Can detect endogenous targets; greater flexibility in dye selection	Higher potential of non-specific binding and cross-reactivity; not compatible for live imaging
	PAINT		No	High	Synthetic dyes	Higher/higher	Can detect endogenous targets; greater flexibility in dye selection; can repeatedly and selectively label the same target	Need appropriate binding affinity for the target structure