Table 2.

The most commonly used linear fluorescence-based super-resolution microscopy modalities to assess ECM components.

Technique	Main Advantages	Main Limitations	ECM and Non-ECM Components Commonly Imaged
SIM	Improved resolution (~100 and 250 nm in the lateral and axial directions) Fast imaging rate Conventional fluorophores can be used Possible use of simultaneous fluorophores	Reduced resolution compared to other super-resolution modalities Vibration must be considered Imaging artifacts due to image processing Sensitive to out-of-focus light Longer processing times when compared to other modalities	Focal adhesions (integrins) Proteoglycans
STED	Confocal-based: fast scanning over small regions No need for specialized fluorophores Laser power tunes resolution Suitable for in vivo applications, where higher temporal resolution is needed No need to computationally reconstruct images	Very high laser intensities required for highest resolutions Vibration must be considered Photobleaching and phototoxicity must be considered Slight improvement in z-resolution when compared to LSCM	Cell-matrix interactions Extracellular fluid flow in the extracellular space Focal adhesions (integrins) Cell-surface receptors Fibronectin
STORM	High spatial resolution (~20–50 nm) Single fluorophores are imaged (important for quantitative imaging) Simple control of fluorophores (not limited to those that can undergo photoswitching, photoconversion, or photoactivation) Lower laser intensity can be used compared to STED (more suitable for in vivo applications)	Low temporal resolution Vibration must be considered Special fluorophores required Phototoxicity associated with multiple imaging/quenching cycles Imaging of regions close to the coverslip Extensive post-acquisition image processing is required Samples can be prone to drift (this can be corrected in most cases)	Actin cytoskeleton Cell-matrix interactions Inorganic ECM fluorophores (bone mineralization) Cell junctions
PALM	High spatial resolution (~20 to 50 nm) Single fluorophores are imaged (important for quantitative imaging) Simplicity in both concept and instrumentation, requiring only a modified widefield fluorescence microscope (to conduct single-molecule imaging) Ability to express fluorescent fusions in adherent cell cultures	Low temporal resolution Vibration must be considered Special fluorophores required Extensive post-acquisition image processing is required Samples can be prone to drift (this can be corrected in most cases)	Focal adhesions (integrins) Cell-surface receptors Cell junctions Mitochondrial proteins
FLIM	Ability to detect changes in the molecular environments of fluorophores Provide information about fluorophore function and behavior (not possible with intensity measurements alone) Do not require the throughput calibration steps that are needed for intensity-based experiments Provides better SNR for weakly fluorescent samples Estimates multiple lifetime components Minimizes the effect of photon scattering in thick layers of sample	Requires costly pulsed lasers Poor performance with high photon count rates or dynamic samples Localized environmental factors, such as autofluorescence or a change in pH, can also shorten the measured fluorescence lifetime and lead to artifacts	Collagens Elastin Extracellular calcium ions Hydroxyapatite deposits

SNR: signal-to-noise ratio; SIM: structured illumination microscopy; STED: stimulated emission depletion; PALM: photoactivated localization microscopy; STORM: stochastic optical reconstruction microscopy; FLIM: fluorescence lifetime imaging microscopy.