Plapon 60xo

For in vivo time-lapse microspcopy studies, fluorescence images, corresponding to mCherry-Rab1b and YFP-Sec24 expression, were both taken at 4 second intervals for 10 min at 0.05% laser power (acquisition time between channels was between 0.5 to 1.5 s, according to the fluorescence intensity of the sample). Fluorescence images corresponding to dual expression of mCherry-Rab1b and GFP-p115 were taken at 6 second intervals for 6 min at 0.05% laser power. Images from triple transfection (mCherry-Rab1b, YFP-Sec24 and SialT2-CFP), were obtained at 30 second intervals for 30 min at 0.05% laser power. One optical Z slice was visualized and imaged using a PLAPON 60XO (1.42 NA) on an Olympus IX81 microscope equipped with a spinning-disk confocal unit (slit mode) DSU (Olympus) and an Orca ER camera (Hamamatsu) under cell^R software control (Olympus Imaging, Center Valley, PA, USA.). During imaging, cells were maintained at 37°C in an incubation chamber (Harvard Instruments).

The NV centre in [100] face bulk diamond was mounted on a typical optically detected magnetic resonance confocal setup, which was synchronized with the microwave bridge by a multichannel pulse generator (Spincore, PBESR-PRO-500). The nitrogen concentration was less than 5 p.p.b. and the abundance of ¹³C was at the natural level of 1.1%. The 532-nm green laser for pumping and phonon sideband fluorescence (650–800 nm) went through the same oil objective (Olympus, PLAPON 60XO, NA 1.42). To preserve the NV centre's longitudinal relaxation time T₁ from laser leakage effects, the pump beam was passed twice through an acousto–optic modulator (ISOMET, power leakage ratio ∼1/1,000) before it went into the objective. We created a solid immersion lens in the diamond around an NV centre (see Supplementary Note 5 and Supplementary Fig. 3). The solid immersion lens increases the PL rate to ∼400 kcounts s⁻¹. The fluorescence intensity was collected by avalanche photodiodes (Perkin Elmer, SPCM-AQRH-14) with a counter card. The adjustable external magnetic field, created by the permanent magnets, was aligned by monitoring the variation of fluorescence counts. Microwave and radio frequency pulses were carried by ultra-broadband CPW with 15 GHz bandwidth.

BmN4 cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, then the cells were permeabilized with 0.3% Triton X-100 in PBS for 5 min. After pre-incubation with blocking buffer [PBS supplemented with 1% BSA (Merck Millipore/Sigma) and 0.1% Triton X-100] at room temperature for 1 h, the cells were incubated with primary antibodies [anti-FLAG antibody (M2; Merck Millipore/Sigma, 1/300), anti-HA antibody (3F10; Merck Millipore/Roche, 1/300), anti-Siwi antibody (1/400), anti-BmAgo3 antibody (1/300), anti-Spn-E antibody (1/250), or anti-DDX6 antibody (1/250)] in blocking buffer at 4 °C overnight. Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 488 donkey anti-rat IgG, Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 647 goat anti-rat IgG, Dylight 488 goat anti-rabbit IgG, and Dylight 594 goat anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) were used for detection. A biotinylated anti-BmAgo3 antibody was used after incubation with the secondary antibody, and the BmAgo3 signal was detected by Cy3-Streptavidin (Jackson ImmunoResearch). Images were captured using an Olympus FV3000 confocal laser scanning system with a ×60 oil immersion objective lens (PLAPON 60XO, NA 1.42; Olympus) and processed with FV31S-SW Viewer software and Adobe Photoshop Elements 10.

Cells grown on coverslips were washed with PBS and fixed with 4% paraformaldehyde in PBS (09154-85; Nacalai) for 15 min at room temperature. Fixed cells were stained with 1 μg/ml Cellstain Hoechst 33342 (23491-52-3; Wako) in Hanks' balanced salt solution for 10 min. After washing twice with PBS, the coverslips were mounted in Prolong Gold antifade reagent (P36930; Thermo Fisher Scientific). The coverslips were observed using a confocal laser microscope (FV1000 IX81; Olympus) with a 60× oil-immersion objective lens (PLAPON60XO; Olympus) and captured with Fluoview software (Olympus). Contrast and brightness adjustment was performed using Fiji (67 (link)). The number of punctate structures was determined using Cellprofiler (68 ).

Imaging was performed on the Olympus FV1200 confocal microscope with GaAsP sensors. A 30×/1.05 silicone immersion objective (UPLSAPO30XS, Olympus) (Figure 1C–D and Figure 2—figure supplement 1B), or a 60×/1.42 oil immersion objective (PLAPON60XO, Olympus) (Figures 2B–D and 3A, Figure 2—figure supplement 1A and Figure 3—figure supplement 1A) was used for scanning specific regions of interest (ROIs). A final voxel size of the image was 0.17 × 0.17 × 0.76 μm³ (Figure 1C–D), 0.11 × 0.11 × 0.45 μm³ (Figure 2B and Figure 2—figure supplement 1A), 0.21 × 0.21 × 0.43 μm³ (Figure 2C–D), 0.10 × 0.10 × 0.45 μm³ (Figure 3A), 0.51 × 0.51 × 0.68 μm³ (Figure 2—figure supplement 1B), and 0.79 × 0.79 × 0.37 μm³ (Figure 3—figure supplement 1A), respectively. Confocal stacks were analyzed with the open-source software ImageJ (National Institute of Health) and Fiji (Schindelin et al., 2012 (link)). Where appropriate, 2D/3D image deconvolution was applied using Diffraction PSF 3D and Parallel Iterative Deconvolution plugins in ImageJ.