Fluorescence fluctuations obtained from Type I-III experiments performed in oocytes with the set of concentrations (<i>i</i>).

<p>The mean fluorescence () and variance () are computed as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#s2" target="_blank">Materials and Methods</a>. The experimental data (black squares) and their corresponding fits (black line) are shown for: (<b>A</b>) 84 images obtained in Type I experiments, fit: ; (<b>B</b>) 21 images obtained in Type II experiments, fit: ; (<b>C</b>) 88 images obtained in Type III experiments, fit: .</p

Simulated fluorescence during a Type 0 experiment in a linescan image.

<p>The fluorescence is computed from the -bound dye distributions of Fig. 6 as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#s2" target="_blank">Materials and Methods</a> Section. (<b>A</b>) and (<b>D</b>) correspond to set <i>(</i><b><i>i</i></b><i>)</i>, (<b>B</b>) and (<b>E</b>) to set (<b><i>ii</i></b>) and (<b>C</b>) and (<b>F</b>) to set (<b><i>iiii</i></b>). (<b>A</b>), (<b>B</b>) and (<b>C</b>) are obtained using equal to the ratio of quantum efficiencies estimated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#pone.0095860-DeYoung1" target="_blank">[22]</a>. (<b>D</b>), (<b>E</b>) and (<b>F</b>) are obtained setting , <i>i.e.</i>, they do not take the –free dye fluorescence into account. In all cases the values of are the ones derived from the fluctuation analyses for the standard illumination power.</p

Behavior of the fluctuation model parameters in the experiments performed under stationary conditions.

<p>Behavior of the fluctuation model parameters in the experiments performed under stationary conditions.</p

Fluorescence fluctuations obtained from Type I–II experiments performed in oocytes with the set of concentrations (<i>ii</i>).

<p>Similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#pone-0095860-g003" target="_blank">Fig. 3</a> but for set (<b><i>ii</i></b>). The experimental data and their corresponding fits are shown for: (<b>A</b>) 84 images obtained in Type I experiments, fit: ; (<b>B</b>) 21 images obtained in Type II experiments, fit: . In this case the results derived from Type III experiments are not shown because no change in fluorescence was observed upon microinjection for this set of concentrations.</p

Dependence of the signal-to-noise ratio on experimentally accessible parameters.

<p>Plots of the signal-to-noise ratio using Eq. 19 with the parameter values determined for Fluo-4 (solid line) and for Fluo-8 (dashed line) as a function of the mean number of dye molecules, (<b>A</b>), the normalized laser intensity, (<b>B</b>) and (<b>C</b>). The vertical dotted lines indicate the range of values and the values of and that correspond to set (<b><i>i</i></b>) at the standard illumination power. See text for more details.</p

Sets of dye and EGTA concentrations.

<p>Sets of dye and EGTA concentrations.</p

Simulated -bound dye concentration during a Type 0 experiment in a linescan image.

<p>sBlurred -bound dye concentration obtained for: (<b>A</b>), (set (<b><i>i</i></b>)); (<b>B</b>) , (set (<b><i>ii</i></b>)); (<b>C</b>) , (set (<b><i>iii</i></b>)). All other parameters are as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#pone-0095860-t004" target="_blank">Table 4</a>. In all the simulations a puff involving the simultaneous opening of 6 IP₃Rs, , occurs at time .</p

Image pre-processing applied to Type I-III experiments performed in <i>Xenopus lævis</i> oocytes.

<p>(<b>A</b>) Typical linescan image obtained with a Type I experiment where dark (cortical granules) and bright fringes (cytosol) are distinguishable. (<b>B</b>) Bright fringes (in white) and dark ones (in black) of the image in (A) identified as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095860#s2" target="_blank">Materials and Methods</a> Section. (<b>C</b>) Final image once the dark fringes have been removed. Once this is done we work with all the pixels of the image without distinguishing their time or spatial coordinates. This pre-processing might not be necessary in other cell types. The color bar represents the fluorescence intensity () both for (<b>A</b>) and (<b>C</b>).</p

Typical row linescan images obtained in oocytes with EGTA and Fluo-4 or Rhod-2 subjected to the same uncaging conditions.

<p>(<b>A</b>) For , , (<b>B</b>) for and , (<b>C</b>) for and . The horizontal and vertical axes correspond to time and space, respectively. The color bar represents the fluorescence intensity (). The white line marks the UV flash. In (<b>A</b>) and (<b>C</b>) several puffs are distinguishable and none can be observed in (<b>B</b>).</p