Du 897 bv

The 3B imaging of Lifeact-mEos3.2 was performed as previously described (Cox et al., 2012 (link)). We used an Olympus IX71 inverted microscope equipped with a 100 × 1.45 numerical aperture (NA) oil objective (Olympus PLAN APO). An internal 1.6× magnification was used to yield a pixel size of 100 nm. An acousto-optic tunable filter (AA Optoelectronic) was used to control the 488-nm laser (Sapphire). The fluorescence signals were acquired using an electron-multiplying charge-coupled device (EMCCD) camera (Andor iXon DU-897 BV). For 3B imaging, Lifeact-mEos3.2 constructs were imaged by a 488-nm laser with 50 ms integration times. The 3B datasets consisted of 200 frames and were corrected for drift.

We used a home-built single molecule microscopy system for all fluorescence imaging experiments and as basis for the combination with an AFM. The system is based on a Zeiss Axiovert 200 inverted epifluorescence microscope equipped with a 100x NA=1.45 oil-immersion Plan-Apochromat TIRFM objective (Olympus). Samples were illuminated in objective-type total internal reflection (TIR) configuration via the epiport using 488 nm light from a solid state laser (Sapphire 200 mW, Coherent), 647 nm light from a Kr⁺ -laser (Innova 301, Coherent), or 532 nm light from a solid state laser (Millennia X, Spectra Physics), with intensities of 3–10 kW/cm². After appropriate filtering, emitted signals were imaged on a back-illuminated, TE-cooled CCD-camera (Andor iXon Du-897 BV). For the precise control of the illumination timings we used acousto-optical modulators (1205 C, Isomet). Timing protocols were generated by an in-house program package implemented in LABVIEW (National Instruments). Illumination times were adjusted to values between 1 and 5 ms. Movies were recorded with a delay in the range of 15 to 300 ms between two consecutive images.

Rat pituitary GH3 cells were cultured at 37 °C in a humidified atmosphere containing 95 % air and 5 % CO₂. The culture medium was DMEM supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. GH3 cells were transfected with small hairpin RNA (shRNA) constructs (Sigma-Aldrich) or the VSVG-mEmerald plasmid (modified from Addgene plasmid #31947) with Lipofectamine 2000 (Invitrogen). For growth hormone secretion assay, cells were transferred to serum free medium 48 h after transfection and incubated for 2 h before ELISA. Live cell confocal images were acquired 48 h after transfection, using spinning disk confocal scan head (CSU-X/M2 N, Yokogawa) attached to an inverted microscope (IX-81, Olympus) and an EMCCD camera (DU897BV, Andor) controlled by Micro-Manager software. Images (512 × 512 pixels, voxel size 0.0946 μm/pixel) were taken every 0.5 s for 400 frames. Live images were analyzed in NIH ImageJ with the MTrackJ plugin. VSVG containing vesicles (10/cell) were randomly selected in 11 Otg1 knockdown cells and 11 scramble shRNAs treated cells. Directionality of each vesicle was defined as its real transport distance divided by linear distance between the start and end positions.