TABLE 1.

An overview of the imaging techniques used to study the structure and composition of cell-cell adhesions with light-based microscopy. SIM: structured illumination microscopy; SMLM: single-molecule localization microscopy; PALM: photoactivated localization microscopy; STORM: stochastic optical reconstruction microscopy; STED: stimulated emission depleted; PLA: proximity ligation assay; FRET: fluorescence resonance energy transfer.

Imaging technique	—	Resolution	Imaging depth	To investigate	Cons	In vivo imaging
Wide-field	—	xy: max 0.2 µm	2–5 µm	Fast and general overview, large field of view	Lower resolution	Possible
				Largely available
				Technically not demanding
Confocal	—	xy: 500–100 nm	1–10 µm	Localization of proteins in and around the intercellular junction complexes, how specific proteins behave in relationship to each other and the cytoskeleton	Lower resolution	Possible
		z: 500 nm		Easily accessible and widely used
Super resolution	SIM	xy: 100–130 nm	Up to 20 µm	Sub-junctional protein organization of adherens junction and connection to cytoskeleton, co-localization experiments, actomyosin around junctions, link microtubuli and tight junctions	Needs some sample preparation optimization; Some technical handling	Possible
		z: 100–350 nm		Can image deeper in cell; easy set-up; conventional fluorescence dyes; 3D possible
	STED	xy: 20–50 nm	Up to 20 µm	Co-localization, connection with actin cytoskeleton	Phototoxicity	Possible
		z: 100–300 nm		High resolution and deep inside the cell	Limited availability in optimal fluorophores
		—		—	Needs sample preparation optimization and some technical handling
	Expansion microscopy	xy: 70 nm; z: 70 nm	Based on imaging technique (confocal or super-resolution)	High resolution with accessible technique	Not yet widely used so effect of spatial changes of junctions during expansion unknown	Possible
	SMLM (including PALM and STORM)	xy: 20–50 nm	200 nm	Single molecules/proteins visible, sub-junctional protein localization, cytoskeleton-junction interface, identification and quantification of the protein organization of junctions, distances between proteins	Limited penetration depth	Possible
		z: 40–100 nm		Very high resolution	Needs sample preparation optimization
		—		—	Some technical handling
Co-localization techniques	PLA	Based on imaging technique used	Based on imaging technique used	Detect and quantify close protein interactions of proteins within 40 nm of each other at the intercellular junction	Based on imaging technique used	Based on imaging technique used
				Analysis with confocal or super resolution microscope
	FRET	Based on imaging technique used	Based on imaging technique used	Protein interactions or co-localization of proteins within 8–10 nm from each other	Based on imaging technique used	Based on imaging technique used
					Construction sensors may be challenging