Measurement of valve formation dynamics at the single-cell level.

<p>(<b>A</b>) The valve formation process can be separated into several phases: an initiation phase, followed by valve formation and morphogenesis, then by valve exocytosis and finally by daughter-cell separation. The two images correspond to the DIC (digital image correlation) and Z-projections of HCK-123 fluorescence (<i>green</i>). In green we can visualize newly-synthesized valves labeled with the silica-associated dye (HCK-123). Scale bar (<i>black</i>): 10 µm. (<b>B</b>) Schematic representation of the microfluidic device used to record valve formation. During valve formation, light intensity, temperature and renewal of the medium were controlled. (<b>C</b>) To quantify HCK-123 fluorescence in individual cells, we developed new software for cell-tracking and local background estimation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046722#pone.0046722.s012" target="_blank">Methods S1</a>). Images at 4 different times are presented. Apparent cell motility is caused by the liquid flow. (<b>D</b>) To precisely quantify fluorescence in each cell we developed a new shape extraction method. The original image was denoised with the TV-means algorithm, leading to a much cleaner image. Then, among the level lines (iso-intensity curves) of the image enclosing the known center of the cell, we considered that the cell boundary was the line with the sharpest contrast (<i>L</i>). This enabled us to compute cell area <i>A(L)</i> as the area of the region enclosed by <i>L</i>), and its width <i>W(L)</i>, defined as the minimum width of a band containing <i>L</i>.