A film is created by evaporating a 20 µL of 5 mg/mL solution of DPPC in chloroform in a glass vial. The film is suspended by incubation with 200 µL vesicle buffer at 60 °C followed by vortexing. The multilamellar dispersion is extruded through 50 nm polycarbonate filter while keeping the extrusion block on a hot plate (65 °C). 20 µL of the extruded vesicles are taken and DNA-lipid conjugates (L-dN241 and L-dN242) are added as shown in Figure . The solution is kept at 65 °C for 30 min. A flow cell is freshly prepared by plasma cleaning and PDMS bonding and 10 µL of the warm DPPC SUVs incorporating two kinds of DNA-lipids is immediately added to the channels and the flow cell is left at room temperature for ~10 min. A mole fraction of the DNA-lipid conjugates is chosen with respect to DPPC such that there are approximately 4 DNAlipid conjugates of each kind on average within a 100 nm×100 nm region of the SLB. After this, the channels are rinsed with 2 mL vesicle buffer. After this, vesicles displaying appropriate complementary DNA-lipid conjugates are added to the flow cell and allowed to bind for ~10 min. The concentration of vesicles is such that there are approximately 500-600 vesicles per FOV and well-separated from one another. It is notable that in absence of the DNA-lipid conjugates in either or both the SLB or vesicles, no binding of vesicles are observed. In order to test whether the DPPC SLBs have any major defects (micrometer-sized cracks, holes, etc), they were incubated with Texas Red-labeled bovine serum albumin (TR-BSA, Invitrogen). If defects were present, TR-BSA bound to the exposed glass surface at the defect sites and the corresponding fluorescence patterns could be imaged by microscope (Figure ). We thoroughly optimized the preparation technique such that we could consistently produce gel phase SLBs free of major defects. Virus-vesicle fusion and Imaging: After tethering the vesicles and then the viruses according to the scheme outlined in Figure , images were taken in 561 nm (magenta) and 488 nm (green) channels at various contiguous non-overlapping locations of the imaging slide. No spots were detected in the green channel images. The pH was dropped by flowing in fusion buffer. After waiting for 30 min, two channel images were taken at the same positions as previous. In the green (QuantiFluor-RNA) channel, new fluorescent spots could be seen overlapping with magenta (membrane) spots corresponding to each fusion event. The explanation for waiting for 30 min is that in previous cryo-ET studies, 30 min was arbitrarily chosen as a time interval which is long enough that the fusion structures reached their final morphologies . In case of nucleozin experiments, the virus particles labeled with DNA-lipid conjugate were incubated in vesicle buffer containing 100 µM nucleozin for 1 h at room temperature. Chromatic aberration correction: Chromatic aberration correction was carried out using the FIJI plugin Detection of Molecules (DoM) version 1.2.4 according to the instructions provided by the developer: . Multiply labeled TetraSpeck beads (Thermo Fisher) were used to carry out channel registration. 1 µL of the commercially available dispersion of 100 nm beads were diluted to 100 µL with Milli-Q water and put on bath sonication for 1 h. A fresh microfluidic flow cell (Figure ) was prepared and functionalized with PLL-PEG. 10 µL of the bead dispersion was added and allowed to adsorb for >10 min. The excess beads were rinsed away. Image stacks were taken consecutively in 488 nm (green) and 561 nm (magenta) channels with exposure and illumination settings such that pixel intensity values of ~10 4 (maximum possible pixel value for a 16 bit image is 65535) were obtained at the center of a particle. Next, the chromatic calibration table containing the displacement vectors across the FOV was calculated. This table was used to correct the images taken in the magenta channel. When we calculated the distances between magenta-green centroid pairs (dMG) of bead signals using ThunderSTORM plugin, we obtained a mean registration error of 8.9±0.4 nm for the whole FOV after chromatic aberration correction. In comparison, the mean registration error without chromatic aberration correction was 76.1±1.4 nm for the whole FOV. ThunderSTORM analysis: The FIJI plugin ThunderSTORM (Version 1.3) (33) was used to superlocalize the signals from individual diffraction limited particles (fluorescent beads, vesicles, or vesicle-virus fusion volumes). A B-Spline wavelet filter (order = 3, scale = 2.0) was used to filter the images. A local maximum algorithm was used to approximate the localization of particles with 2×std peak intensity threshold (2.0*std(Wave.F1)) and 8-neighborhood connectivity. The point spread function was fitted by a weighted least squares method using a 7-pixel fitting radius and initial sigma of 1.6 pixel. Super-localized coordinates were filtered on the basis of pre-defined "sigma" and "uncertainty" values which were kept constant for all comparative analyses to deduce meaningful comparisons between dMG distributions. The super-localized centroids of particles were corrected for chromatic aberration using DoM plugin wherever applicable. Euclidean distance between two super-localized points in magenta (xM, yM) and green channels (xG, yG) is defined as 𝑑 𝑀𝐺 = √(𝑦 𝑀 -𝑦 𝐺 ) 2 + (𝑥 𝑀 -𝑥 𝐺 ) 2 and they were calculated using the "Colocalization" menu.