Usually in a ratio from 1:1 to 1:10. However, for the co-transfection of unlabeled Gag to be meaningful for singlecell or single particle studies, it must be homogenous for all analyzed assembling or budded VLPs. Current interpretations assume incorporation of both types of Gag into VLPs, as well as comparable ratios of both forms within each imaged or producer cell. Here, we develop a methodology to quantify expression levels of unlabeled Gag in single cells using a Torin 1 abmole fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. We then combine this methodology with super-resolution imaging and molecular counting, to resolve the morphology and to estimate the number of Gag proteins in individual Gag clusters. Using this approach we directly study the nanoscale morphology of membrane-bound forming VLPs as a function of unlabeled to labeled Gag ratios in single cells. This allows us to reveal important differences between bulk and single cell measurements when co-transfection procedures are used. The primary requirement for the use of fluorescent labels is that they should not interfere with protein function or spatial organization. Tests of the functionality of a protein fusion are generally designed depending on the specific protein under study. In the case of Gag, its assembly into VLPs constitutes this functional test. Native spatial organization of the protein fusion at the microscale in turn can and should be tested by complementary immunostaining and standard fluorescence imaging. Changes in the spatial organization at the nanoscale, however, are generally not queried because they are difficult to measure. These changes can be substantial, especially for densely packed proteins such as Gag, therefore calling into question the biological relevance of observations using FP tags. PALM imaging combined with molecular counting analysis provides a unique tool to extract nanoscale information on molecular packing and number of molecules per assembling membrane-bound cluster of Gag. We used this method to confirm that unlabeled and labeled Gag proteins are incorporated into assembling VLPs, by showing that the molecular density decreases when unlabeled Gag is co-expressed. Moreover, the superior resolution of this technique allowed us to show that membrane-bound Gag clusters formed from Gag-mEos2 and Gag/ Gag-mEos2 are morphologically indistinguishable, an information until recently only accessible with electron microscopy techniques, which unfortunately lack protein specificity. Interestingly, this is not true for the tandem-dimeric version of Eos or other chimeric Gag fusions. We previously showed that tagging Gag with tdEos leads to a nearly 2-fold increase in the size of membranebound Gag custers. Using the same approach presented here for Gag-mEos2, we observed that the distribution of radii of membrane-bound Gag clusters shifts to a lower average for a mixture of Gag and Gag-tdEos as compared to Gag-tdEos only, indicating that viral assembly is perturbed by the bigger tandem-dimeric label at the level of Gag assembly at the membrane.