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. 2012 Mar 6;7(3):e31566. doi: 10.1371/journal.pone.0031566

Figure 1. Fluorescein-based single-virion assay for the study of internal virion acidification.

Figure 1

A) Loading of virions with pH-sensitive (fluorescein, green) or pH-stable (SRB, red) fluorophores for detection of M2-mediated internal virion acidification (left) or HA-mediated viral membrane fusion (right; 15). Target synthetic bilayers incorporate sialic-acid receptors and a fluorescein pH indicator. B) Selected frames from a representative time-lapse movie monitoring fluorescein-loaded virions at pH 4.5 (t−7, 7 sec before the pH drop; t0, the time of the pH drop; t50, 50 seconds after the pH drop). A subsection (∼1/6) of the imaged ∼70×140 µm area is shown. The green circle marks a single virion analyzed further in C. C) A representative fluorescence intensity-versus-time single-virion trace (green line), the model used to fit the data (inset), best-fit line (black) and fit-derived Gaussian (blue). Fit-derived parameters: tc, Gaussian mean or the time at which the transition is half complete; w, Gaussian width, or 21/2 x the standard deviation of the underlying distribution. Onset time: O = to−(tc−w). Dissipation time: D = 2w, centered at tc.