Functional Ca2+ Channels between Channel Clusters are Necessary for the Propagation of IP3R-Mediated Ca2+ Waves

The specificity and universality of intracellular Ca2+ signals rely on the variety of spatio-temporal patterns that the Ca2+ concentration can display. Ca2+ release into the cytosol through inositol 1,4,5-trisphosphate receptors (IP 3 Rs) is key for this variety. The opening probability of IP3Rs depends on the cytosolic Ca2+ concentration. All of the dynamics are then well described by an excitable system in which the signal propagation depends on the ability of the Ca2+ released through one IP3R to induce the opening of other IP3Rs. In most cell types, IP3Rs are organized in clusters, i.e., the cytosol is a "patchy" excitable system in which the signals can remain localized (i.e., involving the release through one or more IP3Rs in a cluster), or become global depending on the efficiency of the Ca2+ -mediated coupling between clusters. The spatial range over which the signals propagate determines the responses that the cell eventually produces. This points to the importance of understanding the mechanisms that make the propagation possible. Our previous qualitative comparison between experiments and numerical simulations seemed to indicate that Ca2+ release not only occurs within the close vicinity of the clearly identifiable release sites (IP3R clusters) but that there are also functional IP3Rs in between them. In this paper, we present a quantitative comparison between experiments and models that corroborate this preliminary conclusion. This result has implications on how the Ca2+-mediated coupling between clusters works and how it can eventually be disrupted by the different Ca2+ trapping mechanisms.Fil: Piegari, Estefanía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Using two dyes to observe the competition of Ca2+ trapping mechanisms and their effect on intracellular Ca2+ signals

The specificity and universality of intracellular Ca2+ signals rely on the variety of spatio-temporal patterns that the Ca2+ concentration can display. Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) is key for this variety. In this paper, we study how the competition between buffers of different kinetics affects Ca2+ signals that involve Ca2+ release through IP3Rs. The study also provides insight into the underlying spatial distribution of the channels that participate in the signals. Previous works on the effects of Ca2+ buffers have drawn conclusions 'indirectly' by observing the Ca2+-bound dye distributions in the presence of varying concentrations of exogenous buffers and using simulations to interpret the results. In this paper, we make visible the invisible by observing the signals simultaneously with two dyes, Rhod-2 and Fluo-4, each of which plays the role of a slow or fast Ca2+ buffer, respectively. Our observations obtained for different concentrations of Fluo-4 highlight the dual role that fast buffers exert on the dynamics, either reducing the intracluster channel coupling or preventing channel inhibition and allowing the occurrence of relatively long cycles of Ca2+ release. Our experiments also show that signals with relatively high Ca2+ release rates remain localized in the presence of large Rhod-2 concentrations, while the mean speed of the elicited waves increases. We interpret this as a consequence of the more effective uncoupling between IP3R clusters as the slow dye concentration increases. Combining the analysis of the experiments with numerical simulations, we also conclude that Ca2+ release not only occurs within the close vicinity of the centers of the clearly identifiable release sites (IP3R clusters) but there are also functional IP3Rs in between them.Fil: Piegari, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Lopez, Lucía Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Apropiación social de tecnologías libres: una experiencia de monitoreo ambiental participativo y educación ambiental

An experience of articulation between universities, schools and social movements involved in environmental conflicts due to water contamination is systematized. Environmental education and social appropriation of freely developed technologies were promoted for participatory monitoring of water pollution in urban wetlands of the Buenos Aires Metropolitan Area (Argentina). For this purpose, a bioassay based on microalgae was used, coupled with information and communication technologies (ICT) developed by the Community Sensors group (CoSensores). Reflections are presented on the development of free technologies from a perspective of popular education linked to Latin American environmental thought.Se sistematiza una experiencia de articulación entre universidad, escuelas y movimientos sociales involucrados en conflictos ambientales por contaminación del agua. Se promovió la educación ambiental y la apropiación social de tecnologías de desarrollo libre mediante el monitoreo participativo de contaminación del agua en humedales urbanos del Área Metropolitana de Buenos Aires (Argentina). Para ello, se utilizó un bioensayo basado en microalgas, acoplado a tecnologías de la información y la comunicación (TIC), desarrolladas por el grupo de Sensores Comunitarios (CoSensores). Se presentan reflexiones en torno al desarrollo de tecnologías libres desde una perspectiva de educación popular vinculada al pensamiento ambiental latinoamericano

Analysis of the Effect of Soil Roughness in the Forward-Scattering Interference Pattern Using Second-Order Small Perturbation Method Simulations

Soil moisture (SM) is a key geophysical variable that can be estimated at regional scales using remote sensing techniques, by making use of the known relationship between soil reflectivity and the dielectric constant in the microwave regime. In this context, the exploitation of available illuminators of opportunity that currently emit large amounts of power at microwave frequencies (compared to typical synthetic aperture radar systems) is promising. Some published techniques estimate SM by analyzing the interference pattern (IP) between direct and reflected signal as measured by a single antenna (i.e., IP technique). In this letter, a new approach to simulate the IP is proposed, in which the soil roughness is modeled straightforwardly using the second-order small perturbation model. Results illustrate that the ``notch´´ in the VV-polarization IP (related to the Brewster angle) can only be directly observed for very low values of soil rms roughness (s < 0.5 cm). For typical values of soil roughness (s~ 1.2 cm), the notch disappears and only a minimum in the IP is observed near the Brewster angle.Fil: Franco, Mariano Andrés. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: More, Emanuel. Instituto de Altos Estudios Espaciales-mario Gulich; ArgentinaFil: Roitberg, Esteban Gabriel. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Grings, Francisco Matias. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Piegari, Estefanía. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Douna, Vanesa Mariel. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; ArgentinaFil: Perna, Pablo Alejandro. Consejo Nacional de Investigaciónes Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Astronomía y Física del Espacio. - Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Astronomía y Física del Espacio; Argentin

Interaction of geometry and dynamics on the modulation of intracellular calcium signals

Las señales de Ca²⁺ son ubicuas y juegan un rol importante en numerosos procesos fisiológicos como la fertilización o la muerte celular. Su versatilidad se basa en la variedad de comportamientos espacio-temporales que puede mostrar la concentración de calcio intracelular y en que distintos comportamientos pueden inducir respuestas diferentes. La liberación de Ca²⁺ desde el retículo endoplasmático (RE) hacia el citosol a través de receptores de inositol 1,4,5-trifosfato (RIP3s) es una componente clave dentro de los mecanismos de señalización por Ca²⁺. Los RIP3s necesitan unir IP3 y Ca²⁺ para abrirse por lo que la liberación de Ca²⁺ a través de un único RIP3 puede inducir la apertura de otros canales vecinos, mecanismo que se conoce como liberación de calcio inducida por calcio o CICR por las siglas en inglés. Los RIP3s aparentemente se organizan en cúmulos sobre la membrana del RE (clusters). Si el CICR se encuentra limitado a un cluster, las señales pueden permanecer localizadas (puffs). En caso contrario, pueden convertirse en ondas que se propagan entre clusters. Los puffs son los ladrillos de construcción de esas señales más globales que se propagan por toda la célula. Por este motivo, existe un gran interés en la determinación de las propiedades de los puffs, especialmente en vista de la actual controversia sobre la distribución espacial de los RIP3s activables. Las señales de calcio, por otro lado, pueden remodelarse a través de varios mecanismos, entre ellos, el atrapado del Ca²⁺ por parte de buffers. Éstos no sólo disminuyen la concentración de Ca²⁺ libre sino que modifican su distribución espacio-temporal de distintos modos dependiendo de su cinética. Los puffs de Ca²⁺ se han observado en células intactas con técnicas ópticas mostrando que son intrínsecamente estocásticos. La obtención de una imagen correcta de la dinámica de los eventos entonces implica ser capaz de detectar todo el rango de tama~ nos de puffs. Éstos se observan usando indicadores de Ca²⁺ de una longitud de onda visible y buffers exógenos lentos (por ej; EGTA) para disrumpir el CICR entre clusters. Los indicadores de única longitud de onda aumentan su uorescencia al ligar calcio. De esta manera, generan imágenes que dependen fuertemente de su cinética, transporte y propiedades fotofísicas. Por este motivo, es de particular importancia determinar los artefactos que generan las condiciones del experimento. La propia existencia de los puffs depende de que los RIP3s están organizados en cúmulos. Una distribución uniforme de receptores debería dar lugar típicamente a señales propagantes tipo ondas. Los ovocitos de Xenopus laevis son un sistema experimental ventajoso para estudiar estas señales. Durante la fertilización se evoca una onda de Ca²⁺ que se propaga por toda la célula. La fertilización ocurre en el huevo maduro. Se ha observado que, al madurar el ovocito, su RE se configura y la distribución de los RIP3s parece ser más uniforme. Esto tiene un correlato en las características de las señales evocadas en los ovocitos maduros. En esta Tesis se combinaron experimentos, un análisis teórico y simulaciones numéricas para evaluar de qué modo la geometría de la distribución de canales se combina con alguno de los otros aspectos que in uyen sobre la distribución de calcio libre para determinar las características de las señales. El primer aporte fue introducir un método que permite establecer clases equivalentes de experimentos de obtención de imágenes realizados en condiciones diferentes, donde la clase está determinada por la relación señal-ruido que predice el método. Éste también puede utilizarse para estimar el tamaño de las señales más pequeñas que pueden observarse de manera confiable con cada arreglo y para generar imágenes de uorescencia numéricamente con ruido realista. Esta Tesis también contribuyó a estudiar si la presencia del indicador o del EGTA en distintas condiciones experimentales altera la dinámica intracelular de Ca²⁺, en particular, analizar si son capaces de detectar puffs de Ca²⁺ con similar precisión y de qué modo las distintas configuraciones experimentales afectan las propiedades de los puffs observados y la dinámica del Ca²⁺ subyacente. Se pudo determinar que aunque el indicador o el EGTA no alteran la dinámica intracluster del Ca²⁺, el conjunto de eventos observables del experimento es diferente dependiendo del grado de acoplamiento entre clusters. El estudio, por otro lado, permitió inferir que los puffs con mayor liberación de Ca²⁺ provienen de cúmulos donde los RIP3s están muy cerca unos de otros. Para estudiar en más detalle la disrupción del CICR entre clusters vecinos que inducen los buffers lentos, se realizaron experimentos utilizando simultáneamente dos indicadores de calcio de distinta cinética. Utilizando el método introducido pudimos confirmar nuestra conclusión previa sobre cómo se modifica el conjunto de eventos que se evoca en presencia de buffers lentos. Por otro lado, determinamos que la presencia de buffers rápidos da lugar a liberaciones de Ca²⁺ más prolongadas tal vez debido a que reducen el efecto inhibidor del Ca²⁺ sobre los RIP3s. En relación a la razón por la que los buffers lentos disrumpen el acoplamiento entre clusters pudimos concluir que su presencia disminuye el Ca²⁺ basal, aunque no tanto como para explicar la disrupción. El análisis de la diferente distribución espacio-temporal de los buffers lentos y rápidos parecería indicar la presencia de RIP3s en el espacio entre clusters que podrían ser "silenciados" por los buffers lentos evitando así la propagación de las señales entre cúmulos. Finalmente, en la Tesis se han mostrado también resultados preliminares de señales de calcio que se observan en células maduradas, cuyos cambios pueden explicarse en términos de una distribución espacial distinta de RIP3s.Ca²⁺ signals are ubiquitous and play a relevant role in numerous physiological processes as fertilization or cell death. Their versatility relies on the variety of spatiotemporal behaviors that the intracellular calcium concentration can display and that different behaviors can induce different end responses. Ca²⁺ release from the endoplasmic reticulum (ER) into the cytosol through inositol 1,4,5-trisphosphate receptors (IP3Rs) is a key component of the Ca²⁺ signaling toolkit. IP3Rs need to bind IP3 and Ca²⁺ to become open an therefore. Therefore, Ca²⁺ released through one open IP3R can induce the opening of neighboring ones, a mechanism that is knwon as Calcium Induced Calcium Release (CICR). 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. Puffs are the building blocks of global signals that propagate throughout the cell. Thus, there is great interest in determining puff properties, especially in view of the current controversy on the spatial distribution of activatable IP3Rs. Moreover, calcium signals can be remodeled through various mechanisms, such as Ca²⁺ buffers. Buffers not only decrease the concentration of free Ca²⁺ but also modify its spatial and temporal distribution in different ways depending on their kinetics. Ca²⁺ puffs have been observed in intact cells using optical techniques showing that they are intrinsically stochastic. Obtaining a correct picture of their dynamics then entails being able to detect the whole range of puff sizes. Puffs are usually observed using visible single-wavelength dyes and slow exogenous buffers (e.g., EGTA) to disrupt intercluster CICR. Single-wavelength dyes increase their uorescence upon calcium binding producing images that are strongly dependent on their kinetic, transport and photophysical properties. Thus, determining the artifacts that the imaging setting introduces is particularly relevant. The very existence of puffs depends on the fact that IP3Rs are organized in clusters. A uniform distribution of receptors should typically lead to waves-like (propagating) signals. Xenopus laevis oocytes are an advantageous biological system to study these signals since Ca²⁺ release from the ER only occurs through IP3Rs. During fertilization a Ca²⁺ wave that propagates throughout the cell is evoked. Fertilization occurs in the mature egg. It has been observed that maturation of the oocyte induces a reorganization of the ER whereas IP3Rs seem to be distributed more uniformly on its membrane. This has its counterpart on the properties of the Ca²⁺ signals that are evoked in mature oocytes. In this Thesis we have combined experiments, theoretical analyses and numerical simulations to evaluate in which ways the geometry of the channel distributions interacts with the other aspects that affect the intracellular Ca²⁺ dynamics to determine the characteristics of the signals. The first contribution of this Thesis was to introduce a method that establishes equivalence classes of Ca²⁺ imaging experimental conditions, where the class is determined by the signal-to-noise ratio that is predicted by the method. The method can also be used to estimate the smallest signals that can reliably be observed with each experimental setting and to produce numerically generated Ca²⁺ images with realistic noise. The Thesis also contributed to study to what extent experiments performed with different dyes and/or [EGTA] alter the intracellular Ca²⁺ dynamics, in particular, if they are able to detect Ca²⁺ puffs with similar accuracy and in which ways the different experimental conditions affect the observed puff properties or the underlying dynamics of Ca²⁺ itself. We could determine that, although the dye or EGTA does not alter the intra-cluster dynamics, the set of observable events is different depending on the degree of inter-cluster coupling/uncoupling that is induced by the experimental conditions. An analysis of the observations allowed us to show that the events with the largest amounts of released Ca²⁺ came from clusters with densely packed active IP3Rs. To study in more detail the way in which slow buffers disrupt the inter-cluster CICR experiments were performed using two calcium dyes of different kinetics simultaneously. Applying the method introduced previously in the Thesis we could confirm our previous conclusion on how the set of events that is evoked is modified in the presence of slow buffers. We determined that the presence of fast buffers, on the other hand, results in longer periods of Ca²⁺ release perhaps because they reduce the inhibitory effect of Ca²⁺ on IP3Rs. With respect to the way in which the slow buffers disrupt the inter-cluster coupling we could conclude that their presence decreases basal Ca²⁺, although not enough to explain the disruption. The analysis of the different spatial and temporal distribution of Ca²⁺ bound to slow and fast buffers that is observed in the experiments seems to indicate that there are IP3Rs in the space between clusters that could be silenced by the slow buffers thus preventing the propagation of signals between clusters. Finally, in the Thesis we have also shown preliminary results on calcium signals observed in mature cells, whose changes can be explained in terms of a different spatial distribution of IP3Rs with respect to the case of immature oocytes.Fil:Piegari, Estefanía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Changes in Ca2+ removal can mask the effects of geometry during IP3R mediated Ca2+ signals

Calcium (Ca2+) signals are ubiquitous. Most intracellular Ca2+ signals involve the release of Ca2+ from the endoplasmic reticulum (ER) through Inositol 1,4,5-Trisphosphate Receptors (IP3Rs). The non-uniform spatial organization of IP3Rs and the fact that their individual openings are coupled via cytosolic Ca2+ are key factors for the variety of spatio-temporal distributions of the cytosolic [Ca2+] and the versatility of the signals. In this paper we combine experiments performed in untreated and in progesterone-treated Xenopus laevis oocytes and mathematical models to investigate how the interplay between geometry (the IP3R spatial distribution) and dynamics (the processes that characterize the release, transport, and removal of cytosolic Ca2+) affects the resulting signals. Signal propagation looks more continuous and spatially uniform in treated (mature) than in untreated (immature) oocytes. This could be due to the different underlying IP3R spatial distribution that has been observed in both cell types. The models, however, show that the rate of cytosolic Ca2+ removal, which is also different in both cell types, plays a key role affecting the coupling between Ca2+ release sites in such a way that the effect of the underlying IP3R spatial distribution can be modified.Fil: Piegari, Estefanía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Villarruel, Cecilia Liliana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Ca2+ images obtained in different experimental conditions shed light on the spatial distribution of IP3 receptors that underlie Ca2+ puffs

Many intracellular Ca2+ signals involve Ca2+ release from the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors (IP3Rs). The open probability of IP3Rs depends on cytosolic Ca2+ so that these signals involve Ca2+-induced Ca2+-release (CICR). IP3Rs are organized in clusters. The signals they mediate are observed using single-wavelength dyes and, often, a slow Ca2+ buffer (EGTA) is added to disrupt CICR between clusters and keep the signals spatially restricted. It is assumed that the presence of the dye or of EGTA does not alter the intra-cluster Ca2+ dynamics. In this paper we analyze this issue combining experiments and numerical simulations. We compare the properties of local signals known as puffs observed with different dyes and EGTA concentrations. We determine that although the dye or EGTA does not alter the intra-cluster dynamics, the set of observable events is different depending on the degree of inter-cluster uncoupling of the experiment. An analysis of the observations shows that the events that are missed for insufficient inter-cluster uncoupling are those of fastest amplitude growth rate. This agrees with a spatial organization in which the largest amplitude events correspond to clusters with densely packed active IP3Rs.Fil: Piegari, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Sigaut, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentin

Fluorescence fluctuations and equivalence classes of Ca²⁺ imaging experiments.

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

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

Sets of dye and EGTA concentrations.

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