Piegari E et al. (2014), Fluorescence fluctuations and equivalence class...

XB-ART-50815

PLoS One 2014 Jan 01;94:e95860. doi: 10.1371/journal.pone.0095860.

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Fluorescence fluctuations and equivalence classes of Ca²⁺ imaging experiments.

Piegari E , Lopez L , Perez Ipiña E , Ponce Dawson S .

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Ca²⁺ release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a relevant role in numerous physiological processes. IP₃R-mediated Ca²⁺ signals involve Ca²⁺-induced Ca²⁺-release (CICR) whereby Ca²⁺ release through one open IP₃R induces the opening of other channels. IP₃Rs are apparently organized in clusters. The signals can remain localized (i.e., Ca²⁺ puffs) if CICR is limited to one cluster or become waves that propagate between clusters. Ca²⁺ puffs are the building blocks of Ca²⁺ waves. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP₃Rs. Ca²⁺ puffs have been observed in intact cells with optical techniques proving that they are intrinsically Ca²⁺ dyes, slow exogenous buffers (e.g., EGTA) to disrupt inter-cluster CICR and UV-photolyzable caged IP3. Single-wavelength dyes increase their fluorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Determining the artifacts that the imaging setting introduces is particularly relevant when trying to analyze the smallest Ca²⁺ signals. In this paper we introduce a method to estimate the expected signal-to-noise ratio of Ca²⁺ imaging experiments that use single-wavelength dyes. The method is based on the Number and rightness technique. It involves the performance of a series of experiments and their subsequent analysis in terms of a fluorescence fluctuation model with which the model parameters are quantified. Using the model, the expected signal-to-noise ratio is then computed. Equivalence classes between different experimental conditions that produce images with similar signal-to-noise ratios can then be established. The method may also be used to estimate the smallest signals that can reliably be observed with each setting.

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Species referenced: Xenopus laevis

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	Figure 2. Image pre-processing applied to Type I-III experiments performed in Xenopus lævis oocytes.(A) Typical linescan image obtained with a Type I experiment where dark (cortical granules) and bright fringes (cytosol) are distinguishable. (B) Bright fringes (in white) and dark ones (in black) of the image in (A) identified as explained in Materials and Methods Section. (C) 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 (A) and (C).
	Figure 3. Fluorescence fluctuations obtained from Type I-III experiments performed in oocytes with the set of concentrations (i).The mean fluorescence () and variance () are computed as explained in Materials and Methods. The experimental data (black squares) and their corresponding fits (black line) are shown for: (A) 84 images obtained in Type I experiments, fit: ; (B) 21 images obtained in Type II experiments, fit: ; (C) 88 images obtained in Type III experiments, fit: .
	Figure 4. Fluorescence fluctuations obtained from Type I–II experiments performed in oocytes with the set of concentrations (ii).Similar to Fig. 3 but for set (ii). The experimental data and their corresponding fits are shown for: (A) 84 images obtained in Type I experiments, fit: ; (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.
	Figure 5. Fluorescence fluctuations obtained from Type I–II experiments performed in oocytes with the set of concentrations (iii).Similar to Fig. 3 but for set (iii). The experimental data and their corresponding fits are shown for: (A) 84 images obtained in Type I experiments, fit: ; (B) 21 images obtained from Type II experiments, fit: ; (C) 55 images obtained from Type III experiments, fit: .
	Figure 6. Simulated -bound dye concentration during a Type 0 experiment in a linescan image.sBlurred -bound dye concentration obtained for: (A), (set (i)); (B) , (set (ii)); (C) , (set (iii)). All other parameters are as given in Table 4. In all the simulations a puff involving the simultaneous opening of 6 IP3Rs, , occurs at time .
	Figure 7. Simulated fluorescence during a Type 0 experiment in a linescan image.The fluorescence is computed from the -bound dye distributions of Fig. 6 as explained in Materials and Methods Section. (A) and (D) correspond to set (i), (B) and (E) to set (ii) and (C) and (F) to set (iiii). (A), (B) and (C) are obtained using equal to the ratio of quantum efficiencies estimated in [22]. (D), (E) and (F) are obtained setting , i.e., 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.
	Figure 8. Dependence of the signal-to-noise ratio on experimentally accessible parameters.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, (A), the normalized laser intensity, (B) and (C). The vertical dotted lines indicate the range of values and the values of and that correspond to set (i) at the standard illumination power. See text for more details.
	Figure 1. Typical row linescan images obtained in oocytes with EGTA and Fluo-4 or Rhod-2 subjected to the same uncaging conditions.(A) For , , (B) for and , (C) 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 (A) and (C) several puffs are distinguishable and none can be observed in (B).

References [+] :

Berezin, Fluorescence lifetime measurements and biological imaging. 2010, Pubmed