Uplansapo na 1.4 oil

Fluorescence imaging was performed using a two-colour-STED microscopy instrumentation (Abberior Instruments GmbH, Göttingen, Germany). For confocal and STED microscopy, a 100× Olympus UPlanSApo (NA 1.4) oil immersion objective was used. For excitation (λ= 594 nm and λ = 640 nm), a nominal laser power of 20% was applied, and for STED (λ = 775 nm, max. power = 1.2 W), a nominal laser power of 70 % was applied. The pixel size was set to 60 nm (confocal) and 15 nm (non-diffracted), respectively. Minor adjustments of contrast and brightness of the acquired images as well as the Richardson-Lucy deconvolution with a regularisation parameter of 0.001 (stopped after 30 iterations) were carried out with the imspector software (16.1.7098-win64-AIFpgaV3, Abberior Instruments GmbH, Göttingen, Germany).
The 2D object counter and coloc2 plugin in Fiji software (https://fiji.sc/, 64-bit version)were used for the cluster and Mander’s Coefficient Correlation (MCC) analyses, respectively. MCC was performed using the Costes regression threshold. The defined objects were assigned as a cluster being larger than 10⁵ nm² based on STED images with approximately 50 nm resolution. All graphs show the mean values of three independent experiments plotted with the standard error calculated as a two tailed unpaired t-test. A statistical analysis was performed using GraphPad Prism software (version 5).

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).