Imspector software 16

Infected cells were subjected to immunofluorescence staining as the procedure described for spinning-disc imaging. STED microscopy was performed using a 2-color-STED microscope (Abberior instruments GmbH) equipped with a ×100 Olympus UPlanSApo (NA 1.4) oil immersion objective. Nominal laser powers of 20 and 70% were applied for confocal excitation (594 nm) and STED (775 nm, 1.2 W), respectively. Pixel size was set to 60 and 15 nm for confocal and nondiffracted acquisitions, respectively. Minor contrast and brightness adjustments of images and Richardson–Lucy deconvolution (regularization parameter of 10⁻³, stopped after 30 iterations) were carried out using Imspector software 16.1.7098 (Abberior instruments).

Cells that were exposed to fluorescently labeled virions were mounted with Mowiol (Merck), and if indicated, nuclei were stained with Hoechst 33258 (0.5 μg.mL^-1, Thermo Fisher Scientific). Live cell imaging was performed in the continuous presence of viral particles. Both fixed and live samples were imaged with a Leica TCS SP8 confocal microscope equipped with an HC PL APO CS2 63x/1.4 N.A. oil immersion objective. In addition, super-resolution microscopy was used to image ATTO647N-TOSV mounted in Mowiol on PEI-coated coverslips with a 2-color-STED microscope (Abberior instruments GmbH) as described by Kummer and colleagues [51 (link)]. The STED microscope was equipped with an x100 Olympus UPlanSApo (NA 1.4) oil immersion objective, and the pixel size was set to 60 nm (confocal) and 15 nm (non-diffracted), respectively. Minor contrast and brightness adjustments of images and Richardson–Lucy deconvolution (regularization parameter of 10⁻³, stopped after 30 iterations) were carried out using Imspector software 16.1.7098 (Abberior instruments). Images were analyzed with ImageJ v1.53c software (NIH) and the Imspector software (Abberior Instruments GmbH).