*2.4. Super-Resolution Fluorescence Microscopy (SRFM)*

SRFM was performed with a TCS SP8 confocal microscope equipped with Huygens deconvolution suite embedded via a direct interface with LAS X software and GPU arrays (Leica Microsystems, Wetzlar, Germany) at Servei d'Anatomia Patològica from Hospital Sant Joan de Déu (Esplugues de Llobregat, Barcelona, Spain), as previously described [24]. A summary of the protocol is depicted in Figure 4A. Briefly, harvested VLPs were directly loaded onto the microscope slide and adsorbed to the surface of the cover glass after an incubation time of 30 min at RT (Figure 4A). HIV-1 Gag-eGFP VLP preparations were analyzed with 100 X magnification (zoom 5), a line average of 3 and 496 x 496 pixels with HC PL APO CS2 100 X/1.40 OIL objective with the HyVolution2 mode (Leica). Five fields of 13 sections per each biological triplicate were studied in harvested HIV-1 Gag-eGFP VLPs. Deconvolution was performed with the SVI Huygens Professional program and the best resolution strategy (Scientific Volume Imaging B.V., Hilversum, the Netherlands). HIV-1 Gag-eGFP VLP concentration was calculated based on the division of particle number by 3D image volume as previously described [24]. Briefly, direct quantification was performed on deposited HIV-1 Gag-eGFP VLP samples with 23 × 23 × 3 μm in *xyz* from a total loaded volume of 50 μL distributed in 24 × 60 mm under the cover glass and a total height of 34 μm. Assuming complete sample deposition, minimum concentration of Gag-eGFP VLPs was also calculated. PSD analyses were performed using SigmaPlot 12.0 software.
