Table 1.

Pros and cons of the various experimental approaches to study contact sites

Approach	Pros	Cons
Epifluorescence and confocal microscopy	∙Live cell compatible ∙Fluorescent markers of organelles can be readily obtained ∙Can be used to visualize contact-site residents ∙Microscopes are readily available ∙Amenable to high-content approaches	∙Resolution limit of ~250 nm in xy and 500–700 in z is far larger than the size of most contact sites ∙Fixation for immunofluorescence microscopy may introduce artifacts
Super-resolution microscopy	∙Increased resolution over general fluorescence microscopy techniques ∙Some methods are live cell compatible	∙Highly specialized microscopes and accompanying expertise required ∙Fixation is required for some methods and may introduce artifacts
FRET-based reporters	∙Live cell compatible ∙Extremely sensitive to the distance between membranes ∙Can be used to examine contact-site dynamics	∙Technically challenging ∙Careful controls and equimolar expression of the FRET pair are required ∙Requires special microscopy set-up
Irreversible split fluorescence probes	∙Live cell compatible ∙No pre-existing knowledge of the contact site is needed ∙Enables discovery of new contact sites ∙Can be used as synthetic tethers for rescue experiments ∙Compatible with high throughput screening	∙Irreversible binding can stabilize, alter and expand sites of contact ∙Contact-site dynamics cannot be studied
Reversible fluorescence probes	∙Live cell compatible ∙No pre-existing knowledge of the contact site is needed ∙Can be used to examine contact-site dynamics	∙Low-fluorescence intensity of probes can restrict their application
Transmission electron microscopy (TEM)	∙High-resolution imaging of contact-site ultrastructure within the context of a cell ∙Considered the gold standard for the study of contact-site architecture ∙Can be combined with immunostaining to verify bona fide contact-site residents	∙Most useful for abundant contact sites or those whose residents can be readily detected using immuno-EM or CLEM approaches ∙Low throughput ∙Fixation may introduce artifacts
Electron tomography (ET)	∙Provides high-resolution 3D reconstructions of contact-site ultrastructure ∙Fully hydrated and unstained environment reduces artifacts	∙Technically challenging ∙Requires specialized equipment ∙Full 3D reconstructions not possible due to limited tilt range of the sample holder
Scanning electron microscopy (SEM)	∙Enables high-resolution 3D imaging of large specimen volumes	∙Resolving power more limited compared to other EM techniques ∙Time-consuming and computationally-intensive postacquisition processing of large datasets ∙Fixation may introduce artifacts
Cell fractionation	∙Allows for the proteomic and lipidomic analysis of isolated contact sites ∙Enables biochemical characterization of contact-site residents as well as activity	∙Contacts must be able to withstand the fractionation procedure ∙Purity is rarely achieved and contamination by other membranes is common ∙Protocols for most contact sites have not yet been developed
Proximity labeling	∙Does not require pre-existing knowledge of the contact site ∙Can be used to determine the proteome of a contact site when combined with mass spectroscopy ∙Can be used to identify residents of dynamic/transient as well as stable contacts	∙Requires careful controls
Proximity ligation assays (PLA)	∙Can provide quantitative information on contact-site distance and extent of contact ∙Good sensitivity	∙Requires antibodies to the proteins of interest ∙Fixation may introduce artifacts ∙Careful controls are required as changes in PLA signal do not always reflect changes in contact-site extent