Figure 5.

TrIC analysis on a trace with a low signal/noise ratio. (A) 3D trajectory of an FV particle. (B) The corresponding TrIC analysis. (i) The fluorescence intensities of Gag-eGFP and mCherry-Env are shown as a function of time. The red fluorescence intensity is so low that it is impossible to conclude whether fusion has occurred. (ii) The instantaneous velocity determined from the trajectory is plotted with time. The peaks in the plot show that the virus has been taken up by the cell. (iii) The correlation amplitude is above the threshold until ∼7 min and then overlays the automatic negative control. (iv) The relative distance shows a clear colocalization until 4–6 min, when the signals clearly separate. After 7 min, the red particle leaves the ROI used for cross correlation. Due to the very low fluorescence intensity, the resolution for the position of the correlation maximum is poor. (C) Relative trajectory of the Env-labeled envelope with respect to the Gag-capsid during the fusion event.