We used stimulated emission depletion (STED) imaging and expansion microscopy. For STED, 640- and 561-nm diode excitation lasers, a 775-nm STED laser, all pulsed at 40 MHz, and a 100x (Olympus UPlanSApo, 1.4NA) were utilized. A spatial light modulator (Hamamatsu) was used to produce either a doughnut-shaped (2D-STED) or a top-hat (3D-STED) phase mask, shaping different depletion beams without changing the optical setup. To reduce photobleaching at an optimal signal to noise ratios we employed DyMIN® adaptive illumination.
For expansion microscopy, after immunofluorescence staining, gelation, digestion and expansion were performed as described before77 (link). Notably, we extended incubation time in monomer solution to 45 min, gelation time to 2.5 h and digestion was performed overnight. Images were taken with an HC PL APO CS2 40x/1.10 water objective. Expansion microscopy was used for qualitative representation of occludin and ZO-1 morphology.
For expansion microscopy, after immunofluorescence staining, gelation, digestion and expansion were performed as described before77 (link). Notably, we extended incubation time in monomer solution to 45 min, gelation time to 2.5 h and digestion was performed overnight. Images were taken with an HC PL APO CS2 40x/1.10 water objective. Expansion microscopy was used for qualitative representation of occludin and ZO-1 morphology.