Holographic Microscopy Techniques for Multifocus Phase Imaging of Living Cells - Biomedical Optical Phase Microscopy and Nanoscopy - page 109

cell occurred already after 3 h and the cellular impairment progressed rapidly ( Figure 6.9 ,
right panels). After a peak in the cell thickness at 8 h, a necrotic death with cell burst and
cytoplasm release was observed after 20 h ( Figure 6.9A , right panel). These findings are in
accordance with the results of scanning electron microscopy investigations and apotosis/
necrosis assays [75] .
In summary, the results in Figure 6.9 show that Stx1 induces cell swelling followed by
necrotic cell death, which occurred earlier in macrovascular than in microvascular cells. In a
comparative study on the exposure of the same cells to a different toxin (Stx2) that is
described in detail in Ref. [75] , it is demonstrated by similar results from DHM
measurements that this toxin caused no necrotic effects but prevented cells from cell division.
6.5.2 Imaging of Vacuole Formation
The analysis of vacuole formation in cells is illustrated by the results of a DHM study in
which HBMEC cells were treated by the toxin VD90 of EHEC-Vac (for details, see Ref.
[77] ). Therefore, single cells were observed from the time of toxin exposure until cell death.
The same DHM experimental setup used to obtain the results in Figure 6.9 was applied.
Figure 6.10 shows typical stages in the destiny of vacuolated cells as white light images and
quantitative DHM phase images. High-density cell components such as the nuclei and
nucleoli are visible as bright spots in the DHM phase contrast, whereas the vacuoles appear
as dark areas. This indicates a low refractive index of vacuoles near or equal to that of the
cell culture medium and this can be interpreted as a low protein/nucleic acid concentration.
The cell “A” which was fully vacuolated after 21.8 h of exposure to VD90 of EHEC-
Vac ( Figure 6.10A and B ) rounded up during the next 24.5 h ( t 5 46.3 h after exposure)
( Figure 6.10C ), which led to a significant increase in the DHM phase contrast ( Figure 6.10D ).
After an additional time of 25.4 h ( t 5 71.7 h), the cell poured out as shown in the white light
image ( Figure 6.10E ) and evidenced by the decrease in the cell's DHM phase contrast
( Figure 6.10F ). A similar turnover from fully vacuolated status to necrosis was observed for
another cell (cell “B,” Figure 6.10C F , respectively), during an even shorter time period
(25.4 h). This experiment clearly demonstrated that the vacuolated cells undergo necrosis
after treatment with VD 90 of EHEC-Vac. However, probably due to the heterogeneity of
unsynchronized HBMEC population, the time intervals between full vacuolization and
necrosis differ among cells, resulting in a stepwise, rather than a sudden, necrotic death of the
population [77] .
6.5.3 Imaging of Apoptosis and Necrosis
Results from investigations on living pancreatic tumor cells (PaTu 8988 T) demonstrate in
comparison the potentials of DHM for the label-free time-resolved visualization of