1. Set first the intensity threshold that will be used for cell/
nucleus/membrane detection, using the associated fluorescent
channel to yield the best detection in your staining condition
(see Note 23). This could be achieved by manually checking
that the cells/nuclei/membranes are correctly segmented,
using two to three representative pictures.
2. Define host cells from the association of up to three populations: a population of nuclei, a population of cytoplasm and a
population of membranes.
3. Use the different cellular populations to calculate all hostrelated parameters. The total cellular area (expressed in pixels)
and the total number of cells are the most useful parameters to
determine the cellular viability (Fig. 6a).
4. Segment mycobacteria images using a “spot” detection algorithm. Define threshold intensity, i.e. pixels with intensity
above the threshold, as small objects considered as mycobacteria with high intensities compared to the background, and
identify as objects within the cytoplasm region (Fig. 5b).
Remove the background and normalize pixel intensities
between images.
5. Cluster remaining adjacent pixels together to generate a list of
objects. Calculate the area of each object (see Note 24).
6. Step through the list of identified cellular objects to determine
which bacterium is located within each object. Store the data in
arrays or lists.
7. Calculate the total area (expressed in pixels) of intracellular
bacteria labeled (Fig. 5b).
8. Calculate all infection-related parameters. Define “infected”
and “non-infected” populations by using the area of mycobacteria detected within cells as reference criteria. Calculate the
percentage of infected cells.
3.27 Image-Based
Analysis
for Quantification
of Phagosomal
Acidification Using
LysoTracker Reagent
[10]
1. Proceed first to cell and mycobacteria detection and quantification (see steps 1–8 in Subheading 3.26).
2. Segment acidified vacuoles using a “spot” detection algorithm
similar to the one used for mycobacteria. Use “LysoTracker
negative” (non-infected cells) and “LysoTracker positive”
(heat-killed bacteria) controls to set intensity thresholds.
3. Define four distinct cellular populations, “infected” and “noninfected” cells, with or without acidic compartments.
4. Calculate subsequently the co-localization between mycobacteria and acidic vacuoles in infected cells as to define two
mycobacterial populations, “LysoTracker positive” (overlapping acidic region) and “negative” (no overlap).
694
Nathalie Deboosere et al.
nucleus/membrane detection, using the associated fluorescent
channel to yield the best detection in your staining condition
(see Note 23). This could be achieved by manually checking
that the cells/nuclei/membranes are correctly segmented,
using two to three representative pictures.
2. Define host cells from the association of up to three populations: a population of nuclei, a population of cytoplasm and a
population of membranes.
3. Use the different cellular populations to calculate all hostrelated parameters. The total cellular area (expressed in pixels)
and the total number of cells are the most useful parameters to
determine the cellular viability (Fig. 6a).
4. Segment mycobacteria images using a “spot” detection algorithm. Define threshold intensity, i.e. pixels with intensity
above the threshold, as small objects considered as mycobacteria with high intensities compared to the background, and
identify as objects within the cytoplasm region (Fig. 5b).
Remove the background and normalize pixel intensities
between images.
5. Cluster remaining adjacent pixels together to generate a list of
objects. Calculate the area of each object (see Note 24).
6. Step through the list of identified cellular objects to determine
which bacterium is located within each object. Store the data in
arrays or lists.
7. Calculate the total area (expressed in pixels) of intracellular
bacteria labeled (Fig. 5b).
8. Calculate all infection-related parameters. Define “infected”
and “non-infected” populations by using the area of mycobacteria detected within cells as reference criteria. Calculate the
percentage of infected cells.
3.27 Image-Based
Analysis
for Quantification
of Phagosomal
Acidification Using
LysoTracker Reagent
[10]
1. Proceed first to cell and mycobacteria detection and quantification (see steps 1–8 in Subheading 3.26).
2. Segment acidified vacuoles using a “spot” detection algorithm
similar to the one used for mycobacteria. Use “LysoTracker
negative” (non-infected cells) and “LysoTracker positive”
(heat-killed bacteria) controls to set intensity thresholds.
3. Define four distinct cellular populations, “infected” and “noninfected” cells, with or without acidic compartments.
4. Calculate subsequently the co-localization between mycobacteria and acidic vacuoles in infected cells as to define two
mycobacterial populations, “LysoTracker positive” (overlapping acidic region) and “negative” (no overlap).
694
Nathalie Deboosere et al.