ExM/FlipExM samples imaging

Expanded gels were imaged using the same Leica TCS SP8 STED 3X microscope with a pulsed (80 MHz) white-light laser, HyD detectors and spectroscopic detection using a HC PL APO 86 ×/1.20 W motCORR STED (Leica 15506333) water-immersion objective with a correction collar. Each fluorescent channel was imaged in confocal line-sequential mode. For Alexa488, we used 488 nm excitation and 500–560 nm emission range, for Alexa594 we used 594 nm excitation and 605–630 nm emission and Abberior STAR 635P we used 633 nm excitation and 640–750 nm emission. For ExM samples, the size of the field-of-view was in the range of 50–100 µm and had a thickness in the range of 10–20 µm, chosen to cover the whole volume of a dendrite. The dimensions of FlipExM stacks were 20–30 µm in XY and 30–50 µm in Z. In both cases, the pixel size in XY plane was in the range of 60–80 nm and the distance between z-planes was in the range of 150–180 nm. The z-stacks were subjected to a mild deconvolution using Huygens Professional software version 17.10 (Scientific Volume Imaging, The Netherlands) with CMLE (classic maximum likelihood estimation) algorithm with parameters of SNR (Signal-to-Noise Ratio) equal to 15 over 10 iterations.