Figure 3.

(A) Fluorescence lifetime images of a single Legionella cell incubated with FITC-labeled apoLp-III. The bacteria were grown without and with exogenous choline (−choline and +choline, respectively). The color bar placed on the right of the images is the lifetime scale from 2 ns (blue) to 3 ns (red). Images were acquired with a MicroTime 200 confocal lifetime microscope. The samples were excited with a 470 nm pulse of light and emission was collected through a 488 long-pass filter. The longer fluorescence lifetime is indicative of deeper penetration of FITC-apoLp-III into the cell membrane structure. More than 20 individual cells of each type were analyzed and the images show representative effects; (B) Decays of fluorescence intensity following pulse excitation recorded for a single cell of Legionella bacteria treated with FITC-apoLp-III. The bacteria were grown with (red) and without (blue) choline supplementation. The residuals of the best four exponential fits (black line) are shown in the bottom panels. The time of bacterial incubation (1.5 h) with apoLp-III was kept the same for both samples, 470 nm laser light was used for excitation, and the observation was performed through a 488 long wavelength pass filter. The longer fluorescence lifetime is indicative of deeper penetration of FITC-apoLp-III into the cell membrane structure; (C) Normalized histograms of average lifetime distribution taken from pictures presented in (A). For exponential lifetime, deconvolution was applied to build the following histograms: τ1 = 0.53 ns, τ2 = 1.6 ns, τ3 = 4 ns, τ4 = 11 ns in both panels represented by bars with a height proportional to their percentage contribution. Characteristically, bacteria grown without exogenous choline (−choline) incubated with apoLp-III did not show the presence of a relatively long (11 ns) lifetime component.