Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison

Many biological questions require fluorescence microscopy with a resolution beyond the diffractionlimit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulatedEmission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enablean increase in image resolution beyond the classical diffraction-limit. Here, we compare the individualstrengths and weaknesses of each technique by imaging a variety of different subcellular structuresin fixed cells. We chose examples ranging from well separated vesicles to densely packed threedimensional filaments. We used quantitative and correlative analyses to assess the performance ofSIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typicalapplications and to highlight pitfalls associated with the different techniques.