Professional software version 19

The images were acquired using a Leica TCS SP8 STED 3× microscope equipped with a Leica DFC365 FX digital camera with a STED white CS 100 × 1.40 NA oil objective for the optimized overlay of excitation and an STED beam (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). Image capturing was performed using the Leica LAS × 64-bit software package. The acquired images were deconvolved using Huygens Professional software version 19.04 (Scientific Volume Imaging, Hilversum, Netherlands), using the Classic Maximum Likelihood Estimation (CMLE) algorithm, with SNR:07 and 20 iterations. The image analysis was carried out using the Coloc 2 plugin of Fiji software. The significance of each statistical analysis was determined by a Student’s t test. The randomized images for statistical tests were obtained by rotating one of the two channels 90 degrees [33 (link)]. The resolution format was taken at 1024 × 1024 and the corresponding pixel sizes were both 28 nm in x and y. A minimum of five cells were analyzed per each condition.

For confocal imaging, a Leica TCS SP8 confocal microscope was employed with a Leica HC PL APO 63x/1.40 oil CS2 objective. The images of one technical repetition were acquired at the same resolution format and zoom value for accurate image analysis.
STED imaging was performed with a Leica TCS SP8 STED 3× microscope, equipped with a Leica DFC365 FX digital camera with a STED white CS 100 × 1.40 NA oil objective for optimized overlay of excitation and a STED beam (Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany). Image capturing was performed using the Leica LAS ×64-bit software package. The resolution format of the images is 1024 × 1024 and the corresponding pixel sizes are 18 nm in both x and y. The acquired images were deconvolved using Huygens Professional software version 19.04 (14 September 2021, Scientific Volume Imaging, Hilversum, The Netherlands, http://svi.nl, accessed on 20 January 2023), using the CMLE algorithm, with SNR:07 and 20 iterations.