Alteration of cellular thickness of stimulated Caco-2 cells during wound healing.

<p>(<b>A</b>) Averaged profiles <i>S</i> through the cell layer thickness <i>d</i> of control cells and (<b>B</b>) cell layers after treatment with mitomycin c (left) and EGF (right). (<b>C,D</b>) False color-coded pseudo 3D plots of corresponding representative quantitative DHM phase contrast images.</p

Simultaneous monitoring of cellular key characteristics during epithelial wound closure illustrated by results from a single measurement.

<p>(<b>A</b>) The cell covered area <i>S</i>_c 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>B</b>) Over the period of 40 h, the slope of the cellular dry mass Δ<i>DM</i> 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. (<b>C</b>) 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. (<b>D</b>) The cellular volume <i>V</i> of mitomycin c-stimulated cells was constant during the 40 h observation period. In contrast, <i>V</i> of unstimulated control cells and EGF stimulated cells increased continuously and were almost doubled after 40 h. (<b>E</b>) The quotient of total dry mass in the gap and mean dry mass of single cells for each condition reveals the absolute cell number <i>n</i> in the wound. Unstimulated and EGF stimulated cells indicated a marked increase while mitomycin c treatment resulted in an almost constant cell number.</p

Visualization of epithelial wound healing by white light microscopy and DHM.

<p>(<b>A</b>) Conventional white light microscopy is hardly able to visualize outer borders of Caco-2 cells. (<b>B</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).</p

Refractive index, dry mass and cellular volume of stimulated and unstimulated Caco-2 single cells in suspension.

<p>(<b>A–C</b>) Representative quantitative DHM phase images of suspended single Caco-2 cells (coded to 256 gray levels), (<b>A</b>) untreated control cells, (<b>B</b>) after treatment with either epidermal growth factor (EGF) or (<b>C</b>) mitomycin c. (<b>D–F</b>) The me an cellular radius <i>r</i> 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. (<b>G</b>) The refractive index <i>n</i>_cell of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated cells and untreated control cells. (<b>H, I</b>) Dry mass <i>DM</i> and cellular volume <i>V</i> 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; <i>N</i> = 89, ***<i>P</i><0.001 (the numerical data of diagrams G, H, I are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107317#pone.0107317.s001" target="_blank">Table S1</a>).</p

Time constants of key cellular characteristics during epithelial wound closure.

<p>(<b>A</b>) 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>B</b>) The temporal dry mass change of mitomycin c-stimulated cells was significantly decreased as compared to EGF-stimulated and untreated control cells. (<b>C,D</b>) 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; *, <i>P</i><0.05; (the numerical data of the diagrams A–D are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107317#pone.0107317.s002" target="_blank">Table S2</a>).</p

Utilized off-axis setup for digital holographic microscopy (DHM).

<p>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).</p