CLSM analysis of purified PFV Gag-labelled particles. Viruses were produced by transfecting 293T cells with expression plasmids for Env, Pol, RNA and the appropriate C-terminal tagged Gag-AFP and harvested by ultracentrifugation. Subsequently purified virus was incubated on glass cover slips, fixed and the samples covered in Mowiol. (A) Comparison of fluorescence intensities of background subtracted and smoothed pictures (ImageJ plugins). The mean of at least three randomly taken areas of each particle population was determined. Average and Standard Deviation are depicted. (B) Confocal Laser Scanning Microscopy (CLSM) analysis revealed, that only GFP and YFP labelled virus were efficiently detected inside virus capsids. Although all four fluorescent Gag fusion proteins are incorporated into released particles at comparable amounts (compare with Fig. 2), particles made by mCerulean- or mCherry-Gag were only marginally detectable.