Combining expansion microscopy with other super-resolution techniques

Abstract

Expansion microscopy (ExM) was introduced since 2015 and has been fast developed since then. This technique, via physical enlargement of fluorescence carried biological samples, can resolve structures of tens of nanometers with conventional microscopes. Here I discussed the current methods in ExM and influences of different fixation, protease digestion and labelling methods used in ExM. Validation of ExM was also carried out in the work using image registration of microtubule cytoskeletons and the 190 nm periodic structures of β-spectrin ring structures in neurons. Next, the combination of ExM with other super-resolution techniques, e.g. stimulated emission depletion (STED) microscopy was proposed. The centrosome protein CEP152, the primary cilium and microtubule were resolved using expansion STED (ExSTED) microscopy. With the optimized ExSTED microscopy, a sub-10 nm 2D and a sub-50 nm 3D resolution was achieved. Structured illumination microscopy (SIM) was also attempted to image the expanded hydrogels, but severe artifacts were observed. Finally, a tri-functional fluorescent probe was proposed, where a fluorescent dye was linked with a benzyl-guanine and an acrylic acid group. The probe was used to stain a SNAP-tagged nuclear pore protein in cells and used to crosslink proteins to acrylamide-based hydrogel in ExM.