Biomedical Engineering Reference
In-Depth Information
and schizont stage, respectively. Given that the cytoplasm of RBCs consists mainly of Hb,
it is likely that the refractive index is mostly due to the content of Hb. Hb concentration for
individual
Pf
-RBCs can thus be calculated by calibrating from the refractive index of Hb
solutions reported
[34]
. The results are shown in
Figure 12.15B
. The mean values of Hb
concentration are 30.9
6
3.1, 29.3
6
2.4, 23.3
6
2.7, and 18.7
6
2.9 g/dl for healthy RBCs,
ring, trophozoite, and schizont stage, respectively. Because TPM also provides 3D structural
information, cytoplasmic volume of
Pf
-RBCs can be obtained by subtracting the volume of
parasites' vacuole from the volume of whole RBC. The results are shown in
Figure 12.15C
.
Their mean values are 93.1
6
7.9, 88.5
6
11.8, 57.5
6
13.8, and 34.2
6
15.1 fl for healthy
RBCs, ring, trophozoite, and schizont stage, respectively. In addition, total Hb content is
calculated per each
Pf
-RBC. The total amount of Hb in cytoplasmic volume is given by
multiplying Hb concentration and cytoplasmic volume. The results are shown in
Figure 12.15D
. Their mean values are 28.8
6
1.2, 25.9
6
4.2, 13.4
6
3.4, and 6.3
6
2.5 pg
for healthy RBCs, ring, trophozoite, and schizont stage, respectively.
12.5.2 Assessing Light Scattering of Intracellular Organelles in
Single Intact Living Cells
The measurement of a refractive index map by TPM makes it possible to assess the
contribution of individual organelles to the light scattering spectrum. This is because the
refractive index map is an object function that determines the scattering of light. One can
therefore apply TPM for a model-independent method of assessing contributions to the light
scattering from individual organelles in single intact cells. After measuring the 3D index
map of a living cell, it can be modified in such a way so as to eliminate contrast due to a
particular intracellular organelle. By calculating and comparing the light scattering
distributions calculated from the original and modified index maps using the Rytov
approximation, the light scattering contribution from the particular organelle of interest can
be directly calculated. The relative contributions of the nucleus and nucleolus to the
scattering of the entire cell were determined in this way, and the applicability of the
homogeneous spherical model to nonspherical and heterogeneous organelles in forward
scattering was evaluated.
The method is first applied for extracting the scattering distribution of the nucleolus.
The region associated with nucleolus (2) was replaced with refractive index values of the rest
of the nucleus, drawn according to uniform distribution on a pixel-by-pixel basis
(
Figure 12.16B
). This procedure was repeated at every section of different heights in which
the nucleolus border could be clearly determined, leading to generation of a new 3D
tomogram in which the nucleolus is absent. The difference between scattering fields
calculated from the original tomogram and the nucleolus-free tomogram thus provides the
scattering distribution contributed solely by the nucleolus.
Figure 12.16E
shows the scattering
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