Nuclei detection and ring mask calculation by TissueQuest.

<p>Single channel greyscale pictures were processed by TissueQuest software. (A) The nuclei detection was performed according the following parameters (nuclei size: 10, remove small objects: 1, remove weakly stained objects: 1, automated background: no, automated threshold: 5, virtual channel: no, post processing order: remove, merge and Remove labels i) smaller then: 30 μm, larger then: 100 μm, weaker then: 137, stronger then: do not use; use merging rules: no). The detected nuclei are automatically surrounded by a red line. (B) The ring masks were created according the following parameters (interior radius: -0,31 μm, exterior radius; 12,74 μm, use identification cell mask: no, use nucleus mask: no, background threshold: 5; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s001" target="_blank">S1 Fig</a>). The white insert highlights one infected macrophage. The white arrows depict some of the parasites within the ring mask. (C) The area within the ring mask is highlighted in yellow and represents the region of interest in which the screening of parasites can be performed automatically. Representative pictures out of 3 experiments are shown. Bar = 10μm.</p

Detection of intracellular parasites within the ring mask.

<p>Macrophages were cultured in 96-well cultures in the presence (plus <i>L</i>. <i>major</i>, right row) or absence of <i>L</i>. <i>major</i> parasites (w/o <i>L</i>. <i>major</i>, left row). After 72h a DAPI staining was performed to visualize nuclei of the mammalian cells and the DNA rich areas of the parasites simultaneously. Single channel greyscale pictures were processed by TissueQuest software. The ring masks were created as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.g001" target="_blank">Fig 1</a> and material and methods. (A) The ring mask (highlighted in blue) visualizes the cytoplasmatic area of a representative macrophage that was cultured in the absence of parasites. (B) One representative macrophage infected with <i>L</i>. <i>major</i> parasites is shown including the ring mask which is highlighted in blue. Visually determined DAPI positive signals are marked with yellow arrows. For the detection of parasites within the ring masks a virtual channel of the parasite-associated fluorochrome (in our case DAPI) has to be created (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s002" target="_blank">S2C Fig</a>). The following instrument settings were used: Use ring mask: yes, interior radius: -0,31 μm, exterior radius: 12,74 μm, use identification mask: no, use nucleus mask: no, background threshold: 5. (C) One algorithm developed for the detection of weak signals recognizes false positive signals (yellow squares) within the ring mask (the cell shown in A is highlighted in blue) of macrophages that are not infected. (D) False positive signals (yellow squares) are created. The representative (highlighted in blue) macrophage harbors more than 30 parasites, whereas no more than 10 can be visually determined (see B). (E) The algorithm that was developed to detect weak signals recognizes no false positive signals in the ring mask of macrophages that are not infected. (F) All parasites within infected macrophages are recognized (see yellow squares and yellow arrows in (B)). Representative pictures out of 3 experiments are shown. Bar = 10 μm.</p

TissueQuest-based characterization of infected macrophages.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). (A) The infected macrophages were determined automatically by the TissueQuest software. The circle diagrams depict the distribution of infected (black) and not infected macrophages (grey). The infection rate is indicated within the circle diagram. A representative experiment visualizing the infection rate after measuring 310 or 11237 macrophages is shown. (B) The box-plots depict the TissueQuest based quantification of the average number of intracellular parasites per macrophage after analysis of 237 or 8639 macrophages. The gray boxes indicate the range between the first and third quartile. The horizontal lines indicate the median. The whiskers visualize the spread of data. The red bracket highlights the additional information that was generated after measuring 8639 infected macrophages compared to analyzing 237 infected macrophages (***<0.0001). The data shown in (A) and (B) are representative for three experiments. (C) Macrophages were analyzed regarding their number of intracellular parasites and the amount of macrophages (y-axis) harboring the indicated number of parasites (x-axis) are shown. Data are presented in box-blots. The circle diagram highlights the number of macrophages harboring 1–6 (light gray), 7–12 (dark gray) and 12–20 (black) parasites. Data are representative for three experiments. (D) Validation of TissueQuest-based quantification of parasites by real-time PCR was performed. Macrophages were infected with different numbers of parasites per host cell (1:1 and 5:1) and 96 hours post infection the average number of parasites per macrophage (right y-axis) and the number of <i>L</i>. <i>major</i> parasites per β-actin was determined (left y-axis) (*<0,01, *** 0,0001; n = 3; mean +/- SD).</p

Phenotypical analysis of macrophages harboring different numbers of parasites.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). After 72 hours the cells were stained with F4/80^Cy5, CD11b^biotinylated + Streptavidin^AF546. Macrophage nuclei and parasite DNA rich areas were stained with DAPI. (A) The TissueQuest-based analysis of cells and intracellular parasites are presented. A histogram plot depicts the number of parasites within the macrophages (x-axis) and the number of macrophages harboring the indicated (see x-axis) number of <i>Leishmania</i> parasites. Cells within the highlighted gates were further analyzed regarding the expression of CD11b and F4/80. (B) The mean intensities of CD11b or F4/80 are plotted representing macrophages harboring no (#0), one (#1), six (#6) or ten (#10) parasites. Statistical analyses were performed with GraphPad Prism using the nonparametic Mann-Whitney test (p**<0,01, p*<0,05; red horizontal line represents the median). Every single dot represents one individual analyzed nucleated cell. One representative set of data out of two experiments is shown.</p

Assessment of parasite load <i>in situ</i>.

<p>The skin-draining lymph nodes of infected BALB/c mice were removed 20 days after infection. Cryosections were stained with DAPI and analyzed by fluoresence microscopy. (A) One representative region of the infected lymph node is shown. The gray scale image depicts DAPI⁺ host cell nuclei and parasite DNA. (B) An overlay of the detection of nuclei (green), cytoplasm (orange), and parasites (magenta) is shown. (C) Backward gating visualizes cells harboring 3 and (D) 5–10 parasites (highlighted in magenta). E) The graph represents the parasite load (y-axis) within infected cells. The horizontal line represents the median and the arrow bars display the interquartile range. Each dot represents a host cell harboring the indicated (y-axis) amount of parasites.</p