Sp8 falcon microscope

FCS was performed on a Leica SP8 FALCON microscope (as described above) using a HC PL APO CS2 ×40/1.10 water objective with a motorized correction collar. FCS traces (30 s) were measured in U-2 OS cells expressing either HaloTag7 or HaloTag9 in the cytosol (no induction). The cells were labeled with MaP618-CA (150 nM, 2 h) and washed twice for 1 min. Excitation and emission collection was performed as indicated in Supplementary Table 19. Five traces per cell and a total of 36 cells from three biological replicates were measured. Data analysis (correlation and fitting) was performed using the LAS X software (Leica Microsystems) fitting a free three-dimensional diffusion model including a triplet component⁵⁶ . The diffusion amplitude (G(0)) as well as the mean photon counts (PC) over the 30 s trace were used to calculate the molecular brightness (mB, equation (9)). $mB=G\left(0\right)\times \mathrm{PC}$

Synaptic density and synapse composition was assayed in 22 DIV neuronal cell cultures. Cultures were fixed in cold 4% PFA with 4% sucrose for 20 min at RT. Primary antibodies were incubated overnight at 4 °C (Supplementary data 9). Secondary antibodies (Invitrogen) were incubated for 3 h at RT. Hippocampal primary culture: pyramidal cells were selected based on their morphology and confocal images were acquired on a Leica SP8 Falcon microscope using 63X (NA 1.4) with a zoom power of 3 and analyzed with Fiji software. After deconvolution (huygens professional), images were subsequently thresholded, and subsequent analyses were performed by an investigator blind to cell culture treatment¹⁰⁷ .

FCS trajectories were obtained using a Leica SP8 Falcon microscope equipped with a Leica APO 86x/N.A. = 1.20 water-immersion objective with the motCORR automated correction collar, as previously described (Gandin et al., 2021 (link)). Mouse embryonic stem cells (mESCs) in which Halo and SNAP_f tag were inserted into the EIF4E1 and EIF4G1 alleles, respectively, were differentiated into fibroblasts (Gandin et al., 2021 (link)). Twenty-four hours later, cells were labelled with 100 nM JF585-Halo tag ligand for 10 min and 500 nM JF64-6SNAP_f tag ligand for 45 min. A set of two measurements (10 s each) were performed in the cytoplasm and in the nucleus for the indicated conditions in at least 5 cells per condition. FCS and FCCS values were exported and averaged using GraphPad Prism v7.