Measurement of Nanoparticle Uptake by Alveolar Macrophages - Safety of Nanomaterials along Their Lifecycle - page 146

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(a) (b)

(c) (d) (e)

FIGURE 7.7 Method developed for an improved edge detection of macrophages. Cell is
viewed with phase contrast optics. (a) Starting from an automatically found center point
numerous radii are constructed toward the edge of the macrophage. End of each radius is
defined by the maximum alteration of the gradient (derived from the gray values along the
radius) and defines a set of “reference points.” (b) An algorithm starts from neighboring ref-
erence points, travels along the contrasted edge, and demarcates the border of the cell. The
perimeter of the cell is calculated from pixel size; a diagonal step is multiplied by 2 . (c) and
(d) Details of a macrophage with a pseudopodium; (d) edge detection by standard Laplace
filtering, (e) edge detection with a Laplace filter plus detection point method. Magnification:
(a) 400×, (c-e) 1200×.

After differentiation of background, particles, and macrophages, and the correct
recognition of the macrophage's edge, the next step was to track moving macrophages
such as NR8383 macrophages. Therefore, the active ROI needed to be adjusted in
every image and around each single macrophage. Based on the correctly detected
edges, the active ROI was centered around the balance point of the macrophage.
This allowed us to track the highly motile and variously shaped macrophages and to
calculate the migration distance in a properly defined manner (Figure 7.6).

7.3.2.2 Measuring the Uptake of Agglomerated Particles
In principle, the quantification of particle uptake by macrophages would be a simple
task if highly contrasted particles had settled down before macrophages were added.
Under these conditions, each cell would clear a defined region from particles and the
mass of engulfed particles could be deduced from the number of missing particles.
However, the sequence of events is vice versa in cell culture testing as it is in vivo.
Figure 7.6 shows what is to be expected and illustrates the strategy, which has been