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

HyVolution2 SRFM nanoparticle analyses were conducted in HEK 293 and Sf9 supernatants by a simple preparation strategy similar to common cell visualization methods used in confocal microscopy. Images were acquired at 100 X magnification as depicted in Figure 4B, where VLPs correspond to green dots. VLPs presented an elongated structure in the Z plane while a smaller diameter was observed in the XY axis due to the convolution effect of light. A mean VLP diameter of 268 ± 77 nm (*n* = 371) was obtained in HEK 293 samples, whereas an average of 261 ± 104 nm (*n* = 794) was measured for Sf9 supernatants (Figure 4C,D). VLP concentrations were comprised in the range of 109–1010 VLPs/mL in both platforms, representing a 1.7-fold difference for Sf9 over HEK 293-derived VLPs (Table 2). The same acquisition strategy was performed in conditioned medium samples from both cell lines, and no detection of GFP signal was observed (Supplementary materials S1).

**Figure 4.** Super-resolution fluorescence microscopy analysis of HIV-1 Gag-eGFP VLPs produced in HEK 293 and Sf9 cells harvested at 72 hpt and 40 hpi, respectively. (**A**) Sample preparation and analysis workflow and (**B**) Gag-eGFP VLP imaging. (**C**,**D**) PSD analysis of HEK 293 and Sf9 supernatants directly quantified after their addition to the microscope slide, respectively. Negative controls were analyzed in the same conditions as in VLP samples (Supplementary materials S1).


**Table 2.** Quantification of VLP and EV concentrations in transfected HEK 293 and BV infected Sf9 supernatants using different analytical methods.

\* From González-Domínguez et al. [24].
