Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 116

the phase contrast images in Figure 6.12 , the cellular organelles other than the nucleoli
appear only with a marginal contrast. During the cell-rounding process, the maximum phase
contrast is well defined by the center of the resulting spherical structure. Thus, also for this
period of the cell cycle, a reliable detection of the cell center is possible. For the phases of
the cell cycle in which the cells adhere on the substrate with thin morphology, the presented
algorithm is expected to be more efficient than the evaluation of the DHM phase contrast
images for 2D cell tracking by classical edge detection algorithms. During the periods when
the cells adhere, the thickness of the outer cell border areas is # 1 2 m. Consequently,

the cells appear with a low contrast of the boundaries in the DHM phase contrast images
(see Figure 6.12 ) which would affect the robustness of an edge detection-based
determination of the lateral cell position.

μ

In conclusion, the data in Figures 6.12 6.15 demonstrate the applicability of DHM for
quantitative monitoring of the cell division processes during long-term time-lapse
observations. DHM provides an efficient method for automated cell thickness monitoring,
while simultaneously the 2D cell migration trajectories are obtained. Furthermore,
subcellular regions with a higher molecular density than that of the surrounding cell
compartments, such as nucleus and nucleoli, are visible in the phase contrast images.
Possible applications of the method include the minimally invasive quantification of the cell
vitality in the research fields of toxin-mediated cell damage and tumor cell migration
analysis in cancer research.

6.7 Cell Tracking in 3D Environments

The analysis of cell migration processes is an important aspect for the understanding of
morphogenesis and cancer metastasis. Thus, investigations of the applicability of digital
holographic autofocus tracking for 3D cell migration monitoring were performed [83] .
Therefore, human fibrosarcoma tumor cells (HT-1080) within nondenatured collagen fibers
as a 3D tissue model were observed in a Petri dish with a DHM setup as shown in
Figure 6.1 . The preparation of the sample was carried out by following the approach of
Friedl and Brocker [86] . Different from Ref. [86] , a slightly diluted collagen concentration
of 1.28 mg/ml was used to decrease light-scattering effects. In analogy to the experiments
described in Section 6.6 , the temperature was stabilized to 37 C using an incubation
chamber. A series of 61 holograms of three HT-1080 cells within the field of view that
were identified as separated under white light illumination was recorded with fixed
mechanical focus (40 3 magnification microscope lens) for a period of 180 min
( Δt 5 3 min). For each cell, the lateral position is determined from the automatically
refocused phase contrast images as described in Section 6.6 , while the axial sample
displacement is obtained from the autofocus distance (see Section 6.3.4 ). Figure 6.16 shows
the obtained results for t 5 0, 60, 120, and 180 min. Figure 6.16A shows bright-field images