Quantitative imaging of internalized <i>Y. pestis</i>.

<p>(<b>A</b>) Transmission electron microscopy showing <i>Y. pestis</i> CO92 internalized within RAW264.7 macrophages at 2 hr (left panel; scale bar –500 nm) and 8 hr (right panel; scale bar –2 microns) post infection. Some extracellular bacteria (arrow outside the cell) are also visible at 2 hr post infection. Bacteria confined within the vacuoles of the macrophages are shown by a single arrow inside the cell. N - Nucleus. (<b>B</b>) Immunofluorescent staining of RAW264.7 macrophages that were not infected (left panel) or infected with <i>Y</i>. <i>pestis</i> CO92 following pre-treatment with DMSO control (middle panel) or cytochalasin D (right panel). Green - <i>Y</i>. <i>pestis</i> stained with α-F1 antibody, red - phalloidin, blue – nucleus stained with Hoechst dye. Scale bar = 5 µm. (<b>C</b>) Image segmentation of macrophages infected with <i>Y. pestis</i>. Images acquired in UV channel were segmented to mark the boundaries of the nuclei (top left), cell boundaries were segmented on the images based on a 640 nm laser channel (cytoplasm channel, top right), segmentation of total spots (bacteria) for image fields acquired in the 488 nm channel (bottom left), classification of spot candidates (internalized bacteria) based on the attribute values for contrast and spot-to-cell intensity above the limits set by the input parameters (bottom right). The different colored spots in the bottom panels represent individual spots (bacteria) and were used for better visualization. The non-segmented white spots in the bottom right panel represent extracellular bacteria. Scale bar = 20 µm. (<b>D</b>) Dual staining of GFP expressing <i>Y. pestis</i> (Pgm⁻, Pla⁻) in infected RAW264.7 macrophages to quantitate cell-associated and internalized bacteria. RAW264.7 macrophages were infected for 2 hr with 30∶1 MOI of GFP expressing bacteria. After fixation but without permeabilization of the macrophages, the GFP labeled bacteria were immunostained with αF1 antibody followed by staining with Alexa 568 secondary antibody. Graphical representation of number of bacteria expressing GFP alone or dual staining is shown in the Top panel. Bottom panel is the scatter plot representation of single cell analysis of triplicate wells. The mean relative spot (bacteria) intensity in the red channel is plotted against the green channel for all cells with ≥ one classified spot. (<b>E</b>) Enumerating internalized bacteria. Graphical representations of the four output features (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055167#pone.0055167.s008" target="_blank">Table S1</a>) that were collected from the image analysis of RAW264.7 macrophages that were either not infected or infected with <i>Y</i>. <i>pestis</i> following pretreatment with DMSO control or cytochalasin D. The data represents the average of six replicates ± standard deviation, and is representative of three independent experiments.