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

Abstract

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

Figure 1. Microtubules in Drosophila macrophages.

Microtubules were detected with primary and secondary antibodies, the latter coupled to Alexa Fluor 488 for SIM and STED and Alexa Fluor 647 for SMLM. 100% available depletion laser power was used for STED. (a) Single optical section through the microtubule network close to the coverslip. Scale bar, 5 μm. The boxed areas are magnified in (b,c). In the STED image out of focus blur (arrow) and an artefact caused by high laser intensity (arrow head) are visible. (b) Region showing well separated microtubules. Scale bar, 0.5 μm. (c) Region with microtubule bundles. Scale bar, 0.5 μm (d) Results of FWHM measurements of 50 microtubules per super-resolution technique. (e) Representative normalised line profiles for each super-resolution technique. Close to the microtubule, intensity values for SIM can dip into negative values, which is an artefact caused by the SIM reconstruction.

Figure 2. Centrioles in Drosophila primary spermatocytes.

Asterless (Asl) localisation in an orthogonal centriole pair was detected with primary and secondary antibodies, the latter coupled to Alexa Fluor 488 for SIM, STED and SMLM. 100% available depletion laser power was used for STED. Scale bar, 0.5 μm. (a) Single optical section through the centre of the centrioles. (b) Measurements of the barrel widths of 30 centrioles per super-resolution technique. (c) FWHM measurements of 60 centriole walls per super-resolution technique. (d) Representative normalised line profiles for each super-resolution technique.

Figure 3. Actin in COS7 cells.

Actin was detected with phalloidin coupled to Alexa Fluor 488 for SIM and STED and Alexa Fluor 647 for SMLM. 100% available depletion laser power was used for STED. (a) Single optical section through the cell periphery. Boxed areas depict parts of the lamella (b) and the lamellipodium (c). The STED image has been 2D deconvolved. Scale bar, 2 μm. (b) Fine structure of the lamella. Scale bar, 0.5 μm. (c) Fine structure of the lamellipodium. Scale bar, 0.5 μm.

Figure 4. Transport vesicles in COS7 cells.

Sec31A was detected with primary and secondary antibodies, the latter coupled to Alexa Fluor 488 for confocal imaging, SIM and STED and Alexa Fluor 647 for SMLM. 100% available depletion laser power was used for STED. (a,c) Overview of vesicles close to the nucleus in a single optical section. Boxed areas are magnified in (b,d). Scale bar, 5 μm. (b,d) Vesicles of various sizes with a little out of focus background visible as weak, small foci in STED. Scale bar, 0.5 μm.

Figure 5. Trans-Golgi network in COS7 cells.

TGN46 distribution was detected with primary and secondary antibodies, the latter coupled to Alexa Fluor 488 for confocal imaging (a), SIM (b) and STED (c) and Alexa Fluor 647 for SMLM (d). 100% available depletion laser power was used for STED. Shown are single optical sections through part of the TGN. Scale bar, 5 µm. Boxed areas are magnified underneath. Scale bar, 1 µm.

Figure 6. Trans- and cis-Golgi in COS7 cells.

TGN46 distribution (blue) was detected with primary and secondary antibodies, the latter coupled to Alexa Fluor 488 for SIM and STED and Alexa Fluor 647 for SMLM. GM130 distribution (yellow) was detected with primary and secondary antibodies, the latter coupled to Alexa 594 for SIM, TMR for STED and Alexa 532 for SMLM. 50% available depletion laser power was used for STED. (a) Single optical section through part of the Golgi. Boxed areas are magnified in (b–d). Scale bar, 5 μm. (b) Fine structure of the cis- and trans-Golgi. Scale bar, 1 μm. (c) TGN46 localisation. (d) GM130 localisation.