Quantification of single-cell expression by nuclear fluorescence signal.

<p><b>A)</b> For each cell, from a confocal stack, we chose the z slice with the largest nuclear perimeter, then, using ImageJ, drew the nuclear boundary using the ellipse tool, and then used the selection brush tool to correct the boundary (approximated here by the red dashed line). We took the average pixel intensity inside these boundaries. Additional detail is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s007" target="_blank">S7 Fig</a>, which shows the proper identification of the nuclear equatorial plane in actual confocal micrographs. <b>B)</b> Relationship between cytoplasmic and nuclear concentration of fluorescent proteins in intestine cells. GFP or mCherry expression was driven by the <i>daf-21</i> promoter in strains RBW2661 (<i>P_daf-21-GFP</i>) and RBW2642 (<i>P_daf-21-mCherry</i>), respectively. Cytoplasmic and nuclear concentrations of fluorescent protein were measured and plotted as x,y coordinates. Red dots are from mCherry expressing cells; green dots are from GFP expressing cells. We measured the nucleus as in <b>A.</b> To sample the cytoplasm, we measured a nucleus sized area of the cytoplasm in the same z slice as the equatorial plane of the nucleus, with care to avoid autofluorescent granules in the cytoplasm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124289#pone.0124289.s003" target="_blank">S3 Fig</a>). Three randomly selected intestine cells were measured in each of four different animals (12 cells total), two expressing GFP and two expressing mCherry. <b>C)</b> The relationship between cytoplasmic and nuclear concentration of fluorescent proteins in intestine cells of heat shocked animals is shown. GFP or mCherry expression was driven by the <i>hsp-16</i>.<i>2</i> promoter in TJ3001 (<i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>GFP</i>) or TJ3002 (<i>P</i>_{<i>hsp-16</i>.<i>2</i>}-<i>mCherry</i>) animals, respectively. Measurements were made as in <b>B</b>, but cytoplasmic measures were difficult because there was more autofluorescence in heat shocked animals’ cells, and we had to sample a few more cells (15 cells total) to see a decent correlation due to this “noise” in the cytoplasmic measurements. <i>P</i> < 10^–7 for the correlations in <b>B</b> and <b>C</b>.