Uplansapo 100 1.4 na oil immersion objective

HeLa cells were plated on glass coverslips and transfected with PSD95-tRFP (Plasmid #52671, Addgene) and/or EGFP-tagged SynGAP C-terminal constructs EGFP-CCα1 or EGFP-CCPBM plasmids (made in house) were co-transfected into HeLa cells using lipofectamine 2000 according to manufacturer instructions. Cells were then fixed with 4% PFA and washed multiple times with PBS prior to mounting with Prolong Gold with DAPI (P36931, Thermo). Confocal stacks spanning entire cells were obtained using UPlanSApo 100 × 1.4 NA oil-immersion objective mounted on Olympus FV1000 laser-scanning confocal microscope using Nyquist criteria for digital imaging. Maximum intensity projections were used for the analysis. Nuclei of cells were defined by DAPI staining, and the EGFP-CC nuclear localization was calculated as the EGFP (colocalized with nucleus) / EGFP (within entire cell perimeter).

For quantification of the SV density, vesicles were immobilized on FITC-conjugated neutravidin patterns, as described above, and were stained with primary anti-synaptophysin and secondary anti-guinea pig antibodies conjugated to STAR635P. Imaging was performed using an Abberior easy3D STED microscope (Abberior GmbH, Göttingen, Germany) equipped with a UPlanSApo 100 ×, 1.4 NA oil immersion objective (Olympus) and an EMCCD iXon Ultra camera (Andor, Belfast, Northern Ireland, UK). A pulsed 640 nm laser was used for excitation, and an easy3D module 775 nm laser was used for depletion. Images were analyzed using a custom written Matlab script. In brief, the STED images were filtered using a bandpass filter to eliminate background noise and the spots above an empirically-defined threshold were identified. Their number was used to determine the vesicle densities, while their positions were used to measure the inter-vesicular distance. The example STED image presented in Supplementary Fig. S6 was processed by deconvolution, using in-built algorithms in Huygens Essential 4.4 (Scientific Volume Imaging, Hilversum, The Netherlands).

All in vivo experiments were performed at 30°C. Images were acquired using a DeltaVision Elite imaging system (Applied Precision (GE), Issaquah, WA, USA) composed of an inverted microscope (IX-71; Olympus) equipped with a UPlanSApo 100× (1.4 NA) oil immersion objective, InsightSSI solid-state illumination, ultimate focus, and a PCO sCMOS camera.
Excitation and emission were measured with the following filter sets, respectively, and in the indicated order: crGE: CFP 438/24 and 475/24 nm, YFP 513/17 and 548/22 nm, FRET 438/24 nm and 548/22 nm. crGE2.3: FITC: 475/28 and 525/48 nm, A594: 575/25 and 625/45 nm, FRET: 475/28 and 625/45. pHluorin: DAPI: 390/18 and 435/48 nm, FITC: 475/28 and 525/48 nm. For crGE and pHluorin, 32% transmission power and for crGE2.3 2% for the FITC channel and 32% for the A595 and the FRET channel. For the aging experiments, stacks of 3 or 4 images with 0.7 μm spacing were taken, and for other experiments, stacks of 30 images with 0.2 μm spacing were taken at an exposure time of 25 ms for all experiments.