Figure 1.
Utilized off-axis setup for digital holographic microscopy (DHM).
A laser beam is divided by a beam splitter into an object wave, illuminating the specimen through a condenser and an undisturbed reference wave. The object wave interferes with the slightly tilted reference wave on a charge coupled device sensor (off-axis geometry). Morphological changes of the biological specimen lead to changes of the optical path length of the object wave, which are coded in the resulting interference pattern (digital hologram).
Figure 2.
Visualization of epithelial wound healing by white light microscopy and DHM.
(A) Conventional white light microscopy is hardly able to visualize outer borders of Caco-2 cells. (B) Phase contrast images provided by DHM (upper row) enable recognition of cell outlines, which are depicted by segmented DHM phase contrast images (lower row).
Figure 3.
Refractive index, dry mass and cellular volume of stimulated and unstimulated Caco-2 single cells in suspension.
(A–C) Representative quantitative DHM phase images of suspended single Caco-2 cells (coded to 256 gray levels), (A) untreated control cells, (B) after treatment with either epidermal growth factor (EGF) or (C) mitomycin c. (D–F) The me an cellular radius r as assessed by false color coded pseudo 3D representations of quantitative phase images was slightly increased after EGF stimulation and markedly more enhanced after mitomycin c treatment. (G) The refractive index ncell of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated cells and untreated control cells. (H, I) Dry mass DM and cellular volume V of EGF-treated Caco-2 cells were significantly increased as compared to untreated cells but were reduced as compared to mitomycin c-stimulated cells. Data are means ±SE; N = 89, ***P<0.001 (the numerical data of diagrams G, H, I are summarized in Table S1).
Figure 4.
Simultaneous monitoring of cellular key characteristics during epithelial wound closure illustrated by results from a single measurement.
(A) The cell covered area Sc after start of the wound healing assay is markedly decreased after stimulation with mitomycin c as compared to untreated control cells, and EGF-treated cells. (B) Over the period of 40 h, the slope of the cellular dry mass ΔDM of EGF-stimulated cell in the wound gap is slightly decreased in comparison to unstimulated control cells whereas mitomycin c-treated cell only reveal a modest increase in cellular dry mass. (C) The average cell layer thickness as well as temporal thickness increase of mitomycin c-treated and unstimulated control was comparable while EGF-stimulated cell show a dramatically increased cell layer thickness that slightly decreased during the observation period. (D) The cellular volume V of mitomycin c-stimulated cells was constant during the 40 h observation period. In contrast, V of unstimulated control cells and EGF stimulated cells increased continuously and were almost doubled after 40 h. (E) The quotient of total dry mass in the gap and mean dry mass of single cells for each condition reveals the absolute cell number n in the wound. Unstimulated and EGF stimulated cells indicated a marked increase while mitomycin c treatment resulted in an almost constant cell number.
Figure 5.
Time constants of key cellular characteristics during epithelial wound closure.
(A) While the change of cell-covered area per minute of mitomycin c-stimulated cells was significantly diminished as compared to untreated control cells, no significant difference was detected between EGF- and untreated control cells. (B) The temporal dry mass change
of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated and untreated control cells. (C,D) No significant differences in the temporal changes of cell layer thickness
and cellular volume
were detected between EGF or mitomycin c-stimulated cells and untreated control cells. Data are means ±SE; *, P<0.05; (the numerical data of the diagrams A–D are summarized in Table S2).
Figure 6.
Alteration of cellular thickness of stimulated Caco-2 cells during wound healing.
(A) Averaged profiles S through the cell layer thickness d of control cells and (B) cell layers after treatment with mitomycin c (left) and EGF (right). (C,D) False color-coded pseudo 3D plots of corresponding representative quantitative DHM phase contrast images.