Table 1.

Evaluation of PPI techniques in planta involving imaging

Method	Cellular resolution	Dynamics	False positives	False negatives	Applicability	Special features and characteristics
BiFC	●	○	● ●	○ ●	● ●	Suitable for weak/transient PPIs
Split-Luc	○	●	●	●	●  a	Dynamic assembly and disassembly of PPIs can be studied
FRET-APB	●	●	○ ●	●	● ●  b	Fast data acquisition and analysis
FRET-FLIM	● ●	●	○ ●	●	○ ●  c.d	High quality of acquired data
BiFC and FRET	● ●	○ ●	● ●	● ●	○ ●  d,e	Analysis of trimeric complexes
Triple FRET	● ●	○ ●	○ ●	● ●	○ ●  c,d,e	Analysis of trimeric complexes
Homo-FRET	● ●	● ●	○ ●	● ●	○ ●  d	Analysis of higher order complexes Can be combined with FRET-FLIM
FCCS	○ ●	● ●	○	○ ●	○d	Low concentration of POI needed Simultaneous detection of PPI and dynamics
RICS and N&B	○ ●	● ●	○ ●	●	●  c	Simultaneous detection of PPI and dynamics
KSP	○ ●	○ ●	○ ●	○ ●	○ ●  f	Analysis of higher order complexes. Inducible visualization of PPI. Can be added to other methods as intrinsic positive control

‘○’ = ‘no or low’, ‘○ ●’ = ‘medium’, ‘●’ = ‘high’, ‘● ●’ = ‘very high’.

^a Phosphorescence of chlorophyll can mask the signal of luciferase.

^b Not suitable for moving proteins.

^c Data acquisition and analysis can be time consuming.

^d Special technical equipment might be needed,

^e Appropriate controls needed.

^f Rapamycin-induced effects must be considered.