Table 1.

Methods to assess ER–mitochondria contact sites and their advantages/disadvantages.

	Method	Advantages	Disadvantages	Limitations
Fluorescent probe-based	Old FP	Fast to detect contact sites Live compatible Easy to perform Cheap	Overestimation of contacts sites distance Fixation for immunofluorescence can introduce artifacts	Use of genetically encoded probes Resolution limits of common microscopies used to detect the probes signal Lacking structural information
	ddFP and PCA	Easy to detect contact sites Live compatible Useful to detect contact sites dynamics	Not suitable for distance measurement Low fluorescence of probes Fixation for immunofluorescence can introduce artifacts
	FRET	Easy to detect contacts sites Live compatible Sensitive to organelle distances Useful to detect contact sites dynamics	Requires equimolar expression of the two moieties Rapamycin addition Fixation for immunofluorescence can introduce artifacts
	SPLICS	Easy to detect contact sites Live compatible Extremely sensitive to organelle distances Partially useful to detect contact sites dynamics No rapamycin addition	Requires equimolar expression of the two moieties Could be thermodynamically stable Fixation for immunofluorescence can introduce artifacts
Immunodetection probe-based	PLA	Easy to detect contact sites (when players are known) Extremely sensitive to organelle distances	Fixation for immunofluorescence can introduce artifacts Requires antibodies to the proteins of interest PLA partners are not always unequivocally expressed at the contact sites	Availability of specific antibodies Indirect measurements of contact sites due to required chemical reaction to detect the players
	APEX	Biochemical characterization of players at contact sites Combined with proteomic can be used to discover new resident proteins Samples are not contaminated by other organelles	Does not allow to measure the distance at contact sites Does not provide information on spatiotemporal dynamics except for SplitAPEX Fixation can introduce artifacts
Microscope-based	TEM	Morphology structure of contact site within the cells in 2D reconstruction or 3D with ET Can be combined with immunostaining to localize resident proteins at contact sites Measurement of contact sites distances	Fixation can introduce artifacts Does not provide information on spatiotemporal dynamics Useful only for highly abundant contact sites In ET the full 3D reconstructions are not always obtained due to limited tilt range of the sample holder	Information on functionality of contact sites are missing Use of antibodies to detect resident proteins
	SEM	Better quality for morphology structure of contact sites in 3D reconstruction of large specimen volumes Can be combined with immunostaining to localize resident proteins at contact sites	Fixation can introduce artifacts Big challenging Time-consuming and intensive computational processing of data Does not provide information on spatiotemporal dynamics Expensive approach
Biochemical	Cell Fractionation	Biochemical characterization of players at contact sites Combined with proteomic can be useful to discover new resident proteins	Long procedure can introduce biochemical modification altering resident proteins at contact sites Difficulty to isolate pure contact site as contamination are common	Information on quantification, structure and functions of contact sites are not provided Appropriate markers to check other organelles contaminants