Measurement of Nanoparticle Uptake by Alveolar Macrophages - Safety of Nanomaterials along Their Lifecycle - page 139

7.2.5 P artiCle t raCking a nalysis
Supernatants from sedimentation experiments were subjected to particle track-
ing analysis to look for nonagglomerated nanoparticles. A volume of 200 µL was
retrieved from particle-containing media at the end of the observation period and
pipetted onto the stage of a NanoSight Instrument (NanoSight LM10) equipped with
a green laser device (530 nm) and an intensified CCD camera (Andor-DL-658M-
OEM). Particle sizes were analyzed with NTA software 2.1.
7.2.6 g eneral r equirements for the s oftWare d eveloPment
To analyze particle sedimentation and phagocytosis of particles by macrophages
we developed a semiautomated software tool for image analysis. The application
was developed with Visual Studio 2010 based on the .NET-Framework 2.0 and C#
as a programming language. Special emphasis was put on automatic object detec-
tion: Although nanoparticle agglomerates could be detected as dark objects in
phase contrast images in the optical plane, cell bodies of macrophages appeared
encircled by a bright halo with fine pseudopodia, which were only slightly darker
than the background. To identify the complete outer contour of a macrophage the
image software had to consider both types of contrasts, such that special algo-
rithms had to be programmed. In general, the program comprises sequential pro-
cessing steps, such as harmonization of luminance throughout the whole image
stack. Function parameters such as gray levels or filter settings can be adjusted
either manually or automatically by an integrated fuzzy logic. The binarization
of particles and macrophages is followed by an object detection step. Finally,
the number of objects is counted and the particle-covered area is calculated in
user-defined regions. Using well tested settings, the program successfully pro-
cessed complete image stacks and automatically counted particles ingested by
macrophages.
7.3 RESULTS
7.3.1 s edimentation of a gglomerated n anoPartiCles in C ell C ulture m edium
Nanoparticles composed of AlOOH or CeO 2 , when exposed to physiological flu-
ids or cell culture medium, tend to readily agglomerate. This is mainly, though
not exclusively, due to the breakdown of the zeta-potential upon ion binding to
charged particle surfaces, such that van der Waals forces become dominant. As
shown in Figure 7.1, these agglomerates are up to several micrometers large and,
therefore, will undergo gravitational settling (cf. Teeguarden et al. 2007). The next
chapters will describe how the sedimentation and uptake of these agglomerates can
be quantified.
7.3.1.1 Quantity of Sedimented Agglomerates
In a first step we simulated more than 100 sedimentation scenarios using different
shapes, sizes, and agglomeration rates of particles. From these results we defined
a sedimentation model, which described the time-dependent change in the single