Imspector

Multi-channel 3D image series were acquired with a Perkin Elmer Ultra VIEW VoX 3D spinning disk confocal microscope (SDCM) using a 100x oil immersion objective (NA 1.4) (Perkin Elmer), with a z-spacing of 200 nm. Images were recorded in the 405, 488, 561 and 640 nm channels. Images of RNA FISH samples were acquired with a Leica SP8 DLS laser scanning confocal using a 63x oil immersion objective (NA 1.4) (Leica, Wetzlar, Germany), with a z-spacing of 300 nm. Stimulated emission depletion (STED) imaging was performed with a λ = 775 nm STED system (Abberior Instruments GmbH, Göttingen, Germany), using a 100x Olympus UPlanSApo (NA 1.4) oil immersion objective. Images were acquired using the 590 and 640 nm excitation laser lines. Nominal STED laser power was set to 80% of the maximal power of 1250 mW with 20-30µs pixel dwell time and 20 nm pixel size. For 3D STED data 60% of the STED laser power was used for fluorescence depletion in the Z-axis and RESCue illumination scheme was used to minimize bleaching. Sampling frequency was 30 nm in all three axis (xyz). All STED images shown (except 3D STED images) were linearly deconvolved with a Lorentzian function (fwhm 50 nm) using the software Imspector (Abberior Instruments GmbH).

The tissue sections of Clta-reporter and WT animals were prepared as already described in the immunofluorescence section. STED microscopy was performed on a home-built setup [72 (link)] with the following settings: eGFP was excited at 483 nm with an average power of 12 μW and depleted at 595 nm with 21 mW average power at the back aperture of the objective lens (PL APO, 1.3 numerical aperture, glycerol immersion; Leica, Wetzlar, Germany). STED images were recorded with 30 × 30 nm pixel size and 20 μs pixel dwell-time. STED and corresponding confocal images were smoothed with a low-pass gauss filter in the acquisition software Imspector (Abberior Instruments). Images of Clta-reporter and WT mice were recorded with the same settings.

3.33 × 10³ SNAP.OR3 expressing cells were seeded on 15-well µ-Slides Angiogenesis (ibidi) and infected as described above. Prior to fixation, cells were incubated with 2 µM SNAP-Cell 647-SiR (New England Biolabs) for 30 min at 37°C, washed three times and fixed with 4% PFA (15 min). STED microscopy was performed using a 775 nm STED system (Abberior Instruments GmbH, Germany) equipped with a 100 x oil immersion objective (NA 1.4; Olympus UPlanSApo). STED Images were acquired using the 590 and 640 nm excitation laser lines while the 405 and 488 laser lines were acquired in confocal mode. Nominal STED laser power was set to 80% of the maximal power (1250 mW) with 20 µs pixel dwell time and 15 nm pixel size. STED Images were linearly deconvolved with a Lorentzian function (FWHM 50 nm) using the Richardson-Lucy algorithm and the software Imspector (Abberior Instruments GmbH).

Representative still images or single frames of image sequences were chosen. Super resolution STED images were deconvolved with a Lorentzian function (full width half maximum = 60 nm) using the software Imspector (Abberior Instruments GmbH, Göttingen, Germany). For all images shown, the camera offset value was subtracted and the contrast and brightness were adapted for optimal display of the image. To eliminate background noise, a 0.5 px median filter was applied to all SDC and TIRF images. Images are shown in greyscale or pseudo colors. In the latter case, the Fire and Green Fire Blue lookup tables (LUTs) were used for SDC and TIRF images, respectively, while different channels of super resolution STED images are shown with the LUTs Red Hot (referred to as ‘red’) and Green Fire Blue (referred to as ‘cyan’).

Images were linearly deconvolved with a Lorentzian function (full width half maximum = 60 nm) using the software Imspector (Abberior Instruments GmbH, Göttingen, Germany). Line profiles of selected assembly sites were generated manually in FIJI (RRID:SCR_002285) (Schindelin et al., 2012 (link)). The intensity values in the Gag and Env channel were exported to Excel. To align the line profiles of different assembly sites, the Gag intensity peak of an assembly site was set to x = 0 nm and the corresponding Env intensity profile was adjusted accordingly. Intensity values were exported to GraphPad Prism (GraphPad Software, Inc., La Jolla, USA; RRID:SCR_002798), normalized (smallest value = 0, highest value = 100) and the average normalized fluorescence intensities ± SEM were plotted.