Mirror-enhanced super-resolution microscopy.

MEANS sample preparation In MEANS microscopy, a piece of mirror is employed in place of the microscope slide to provide the interference. Custom-made, first-surface mirrors were used (China Daheng Group, Beijing, China). The mirror should be a first-surface mirror, with a protective SiO 2 coating, and an adjustable thickness (50, 100, 150 and 200 nm were tested), so that the constructive interference with a high NA objective can occur within the specimen. Due to the existence of the silica layer, cells can grow normally on the mirror surface. A coverslip can be applied to seal the specimen. As shown in Supplementary Scheme 1b , we have a custom-made mirror holder that is the same size as a microscope slide so that the mirror-backed specimen can be placed easily on any commercial confocal microscope.

Cellular sample preparation

Vero cells (CCL-81, ATCC, Manassas, VA, USA) were maintained in high-glucose DMEM (Lonza, Basel, Switzerland) with 10% fetal bovine serum (GE Healthcare Life Sciences HyClone, Logan, UT, USA), 100 U ml −1 penicillin and 100 μg ml −1 streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The human respiratory syncytial virus (hRSV) strain A2 (VR-1544, ATCC, Manassas, VA, USA) was propagated in HEp-2 cells (CCL-23, ATCC) at a titer of 1 × 10 6 p.f.u. ml −1 Cells were plated the day before infection at 25% confluency. Cells were infected by removing the media, washing with phosphate-buffered saline (PBS) (without Ca 2+ and Mg 2+ ions, Lonza, Basel, Switzerland), adding virus at a multiplicity of infection (MOI) of 1, and incubating the cells for 1 h at 37 °C. After adsorption, fresh medium was added to the inoculum. The cells were grown on top of a first-surface mirror coated with an SiO 2 protective layer. Vero cells were fixed with either 4% paraformaldehyde (VWR, Radnor, PA, USA) in PBS for 10 min at room temperature and then permeabilized with 0.2% Triton X-100 (for nuclear pore complex immunofluorescent staining, VWR) or with 100% ice-cold methanol for 10 min at −20 °C and then permeabilized with 100% ice-cold acetone for 2 min at −20 °C (for microtubule immunofluorescent staining, VWR). Nonspecific antibody binding was blocked with 5% bovine serum albumin (EMD Millipore, Darmstadt, Germany) in PBS for 30 min at 37 °C. The cells were then incubated with a primary antibody for 30 min at 37 °C, washed twice in PBS, incubated with a secondary antibody for 30 min at 37 °C, washed twice in PBS and mounted in a mixture of Mowiol 4-88 (Sigma Aldrich, St Louis, MO, USA) and DABCO (VWR) 19 . The primary antibodies used were rabbit anti-alpha tubulin (polyclonal IgG, catalog: ab18251, Abcam, Cambridge, MA, USA) and mouse anti-nuclear pore complex (NPC) proteins that contain FXFG repeats (monoclonal IgG, catalog: ab24609, Abcam). The secondary antibodies used were goat anti-rabbit DyLight 650 (Thermo Fisher Scientific, Waltham, MA, USA) and donkey anti-mouse AlexaFluor 488 (Thermo Fisher Scientific). Spin capture of RSV filaments on glass To capture single hRSV filamentous virions on glass, hRSV A2 was propagated in HEp-2 cells at an MOI of 0.1. At 4 days post infection, the cell-associated and supernatant fractions were scraped, freeze-thawed and spun through 5 and 0.45 μm pore-size centrifugal filters (EMD Millipore) at 5000 × g and 4 °C for 4 and 1 min, respectively. The fraction between 0.45 and 5 μm in diameter was collected and immobilized onto a poly- l -lysine (Sigma Aldrich)-coated, first-surface mirror or cover glass by adsorption of 500 μl of filtered virus for 2 h at 4 °C. The immobilized virions were fixed using 4% paraformaldehyde and were immunofluorescently stained according to the aforementioned protocol. The antibodies used were anti-RSV F monocolonal (palivizumab, MedImmune, Gaithersburg, MD, USA) and anti-RSV N monoclonal (monoclonal IgG1, ab22501, Abcam). Coverslips were mounted in a mixture of Mowiol and DABCO (VWR) 19 . MEANS-STED MEANS-STED imaging was performed with a Leica TCS SP8 STED 3X system equipped with a white light laser as excitation and 592 and 660 nm for STED depletion. The HyD detector and 100 × oil-immersion objective (NA 1.4) were employed. Time-gated detection was also used 20 , in which the AlexaFluor-488 detection is delayed 0.5 ns, whereas the Cy5 detection is delayed 1 ns.

Supplementary Material Supplementary Information Click here for additional data file. Supplementary Video S1 Click here for additional data file. Supplementary Video S2 Click here for additional data file.