Biomedical Engineering Reference
In-Depth Information
of the sample under white light illumination. The corresponding digital holographic phase
contrast images are shown in
Figure 6.16B and C
.
The tracked cells are denoted as A, B, and C. The dotted white squares in the phase
contrast images mark the applied ROIs that were used for holographic autofocusing and 2D
tracking. In the white light images, cells B and C are only slightly defocused during the
experiment. Cell A is located in a different layer and thus appears only marginally visible
due to a large defocus. In
Figure 6.16A
, the ROI for digital holographic autofocusing
tracking was set to cell C, which appears sharply focused in the reconstructed quantitative
phase contrasts images and even subcellular structures such as the nucleoli are visible. As
the axial position of cell B is close to the layer in which cell C is located, this cell is also
displayed with comparable image sharpness. In contrast, cell A is far out of focus. In
Figure 6.16C
, the ROI was set to cell A. After digital holographic autofocusing, cell A is
sharply resolved in each phase distribution and cellular motions as well as deformations and
thickness changes during the process of migration become clearly visible.
Figure 6.16D and
E
shows the resulting temporal dependencies of the relative
z
positions
Δg
as well as the
corresponding (lateral)
x
,
y
coordinates for all three cells A, B, and C. The 3D trajectories
resulting from the combination of the data in
Figure 6.16D and E
are plotted in
Figure 6.16F
.In
Figure 6.16D
F
, data with phase-unwrapping errors were not considered.
For cells B and C, 74% (45 of 61 phase images) were faultlessly determined. For cell A,
77% (47 of 61 images) was obtained. The reason for different percentages of phase
distributions without unwrapping errors is the mutual influence by unfocused structures
outside the ROI. Cells B and C are stationary in position and shape. In contrast, cell A is
located in a layer approximately 80
m below cells B and C and migrates within the
collagen. During the observed migration process through the network of collagen fibers, the
cell undergoes permanent shape changes in a multistep cascade of coordinated adhesion and
contraction which can clearly be observed in
Figure 6.16C
.
μ
In summary, the results in
Figure 6.16
illustrate the ability of DHM for 3D particle and cell
tracking. However, it has to be mentioned that due to the underlying principles of the
presented DHM configurations integral information is obtained. Thus, specimens in
different planes at the same lateral position with axial distances near the depth of field of
the applied imaging system cause diffraction patterns. This can lead to misinterpretations in
Time-dependent digital holographic 3D migration monitoring of human fibrosarcoma HT-1080
tumor cells in collagen. (A) Bright-field images under white light illumination; (B) quantitative
digital holographic phase contrast images with autofocus tracking ROI set to cell C, as marked
with a dashed box; (C) quantitative digital holographic phase contrast images with autofocus
tracking ROI set to cell A, as marked with a dashed box; (D) temporal dependency of the axial
positions of cells A, B, and C; (E) time-resolved lateral x,y tracking of cells A, B, and C; and
(F) resulting 3D trajectories. Source: Modified from Ref.
[83]
.
Search WWH ::
Custom Search