Table 1.

Methods for Imaging Molecular Dynamics and Interactions in Living Cells

Technique	What It Measures	Advantages	Disadvantages	Refs.
FRAP FLIP, iFRAP	Protein mobility and  compartmentalization	Standard method on most  LSCM systems	Complex decay kinetics,  photodamage artifacts	12, 38, 39,  10, 46, 47
Photoactivation		Allows user to switch on FP,  molecular highlighters with  high contrast	Limited fluorophores  available, complex kinetics	29, 46
FCS	Protein mobility and  complex formation	Requires low FP  concentrations, high  sensitivity, spatial, temporal  resolution	Difficult to implement,  complex decay kinetics,  sensitive to photobleaching	55, 56, 57
FRET SBT  correction	FRET quantifies spatial  relationships between  fluorophores (<80Å) arising from protein  interactions and protein  conformation.	Computer algorithms  available, can follow  specific interactions with  time	Requires careful controls to  establish SBT correction.  Values vary with algorithm  and detection equipment.	71–74, 78
pbFRET		Easiest and most accurate  way to quantify FRET	Endpoint assay, cell  movement artifacts	81, 82
FLIM-FRET	Sensitive to different local  microenvironments	Independent of intensity; can  provide most rigorous  information about  interactions.	Complex, multicomponent  lifetimes difficult to interpret  and may be additionally  affected by cell  environment	86 – 90