Media 4: Enhanced quantitative phase imaging in self-interference digital holographic microscopy using an electrically focus tunable lens

Originally published in Biomedical Optics Express on 01 December 2014 (boe-5-12-4213

Media 2: Enhanced quantitative phase imaging in self-interference digital holographic microscopy using an electrically focus tunable lens

Originally published in Biomedical Optics Express on 01 December 2014 (boe-5-12-4213

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

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

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

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

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

Calcium flux experiments.

<p>Calcium flux experiments using the dye Fluo-4 in combination with structured illumination fluorescence microscopy: Representative microscopy images of the cells before and after the stimulation with the different treatments. From each experiment images with the maximum fluorescence signal after stimulation are shown. (a) Panel with the controls. (b) Panel with the nanoformulations. (c) Fluorescence intensity over time after stimulation with the different treatments (Scale bar = 200 μM). NC: nanocapsules (images in (a) and (b) have been contrast enhanced for better visualization in the print version).</p

Fluorescence microscopy.

<p>Representative images of MDCK cells with actin or ZO-1 staining. (Nuclei: blue, ZO-1: green, Actin: red, NC: nanocapsules) (images have been contrast enhanced for better visualization in the print version)”.</p