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Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

Ríos E, Stern MD, González A, Pizarro G, Shirokova N - J. Gen. Physiol. (1999)

Bottom Line: Proc.Natl.Real sparks differ from simulated ones mainly in having greater width.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612, USA. erios@rush.edu

ABSTRACT
An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

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The system's PSF. (A) Fluorescence intensity in xy sections of the image of a 0.1 μm bead, plotted as a function of distance in the plane of the section and value of the axial variable z. For each xy section (or discrete z value) data were reduced to a function of the radial distance to the center of the bead  by averaging fluorescence in concentric rings of increasing radius. (B) Average fluorescence per pixel as a function of axial distance, at the center of the bead (r < 0.14, •) and in the ring between 0.285 and 0.428 μm (○). The continuous curves are gaussian fits with parameters given in the text. (C) fluorescence versus r, for three ranges of z, and the corresponding gaussian fits (ranges and parameter values given in text).
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Figure 1: The system's PSF. (A) Fluorescence intensity in xy sections of the image of a 0.1 μm bead, plotted as a function of distance in the plane of the section and value of the axial variable z. For each xy section (or discrete z value) data were reduced to a function of the radial distance to the center of the bead by averaging fluorescence in concentric rings of increasing radius. (B) Average fluorescence per pixel as a function of axial distance, at the center of the bead (r < 0.14, •) and in the ring between 0.285 and 0.428 μm (○). The continuous curves are gaussian fits with parameters given in the text. (C) fluorescence versus r, for three ranges of z, and the corresponding gaussian fits (ranges and parameter values given in text).

Mentions: The fluorescence averaged in this way is represented versus r and z in Fig. 1 A. The dependence could be fitted as the product of two gaussians: P . The two-gaussian description, which is used later in the analysis, is documented in B and C. Fig. 1 B contains data as a function of the axial coordinate z. The fluorescence values represented are averages within each section, over a 0.1425-μm circle centered on the line (•), or in a ring region 0.285 < r < 0.4275 μm (○). The lines are best fit gaussians with , respectively, indicating some deviation from the product of gaussians at high values of r. In Fig. 1 C, the fluorescence is represented as a function of distance r at constant z. The three sets of data are from the central section (/z/ < 0.18 μm; •), an intermediate region (0.18 < /z/ < 0.54 μm; dotted symbols), and an outlying region (0.72 < /z/ < 0.98 μm; ○). The best fit gaussians shown have very similar spread . In the analysis of data, we neglect the deviations and approximate the PSF as a product of a gaussian function of r, with (corresponding to a full width at half magnitude [FWHM] of 0.47 μm) and a gaussian function of z, with (FWHM of 1.44 μm). Similar values were obtained with a second C-apochromat 1.2 N.A. objective in our microscope, and with either objective in a LSM 410 (Carl Zeiss, Inc.) confocal scanner.


Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

Ríos E, Stern MD, González A, Pizarro G, Shirokova N - J. Gen. Physiol. (1999)

The system's PSF. (A) Fluorescence intensity in xy sections of the image of a 0.1 μm bead, plotted as a function of distance in the plane of the section and value of the axial variable z. For each xy section (or discrete z value) data were reduced to a function of the radial distance to the center of the bead  by averaging fluorescence in concentric rings of increasing radius. (B) Average fluorescence per pixel as a function of axial distance, at the center of the bead (r < 0.14, •) and in the ring between 0.285 and 0.428 μm (○). The continuous curves are gaussian fits with parameters given in the text. (C) fluorescence versus r, for three ranges of z, and the corresponding gaussian fits (ranges and parameter values given in text).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229636&req=5

Figure 1: The system's PSF. (A) Fluorescence intensity in xy sections of the image of a 0.1 μm bead, plotted as a function of distance in the plane of the section and value of the axial variable z. For each xy section (or discrete z value) data were reduced to a function of the radial distance to the center of the bead by averaging fluorescence in concentric rings of increasing radius. (B) Average fluorescence per pixel as a function of axial distance, at the center of the bead (r < 0.14, •) and in the ring between 0.285 and 0.428 μm (○). The continuous curves are gaussian fits with parameters given in the text. (C) fluorescence versus r, for three ranges of z, and the corresponding gaussian fits (ranges and parameter values given in text).
Mentions: The fluorescence averaged in this way is represented versus r and z in Fig. 1 A. The dependence could be fitted as the product of two gaussians: P . The two-gaussian description, which is used later in the analysis, is documented in B and C. Fig. 1 B contains data as a function of the axial coordinate z. The fluorescence values represented are averages within each section, over a 0.1425-μm circle centered on the line (•), or in a ring region 0.285 < r < 0.4275 μm (○). The lines are best fit gaussians with , respectively, indicating some deviation from the product of gaussians at high values of r. In Fig. 1 C, the fluorescence is represented as a function of distance r at constant z. The three sets of data are from the central section (/z/ < 0.18 μm; •), an intermediate region (0.18 < /z/ < 0.54 μm; dotted symbols), and an outlying region (0.72 < /z/ < 0.98 μm; ○). The best fit gaussians shown have very similar spread . In the analysis of data, we neglect the deviations and approximate the PSF as a product of a gaussian function of r, with (corresponding to a full width at half magnitude [FWHM] of 0.47 μm) and a gaussian function of z, with (FWHM of 1.44 μm). Similar values were obtained with a second C-apochromat 1.2 N.A. objective in our microscope, and with either objective in a LSM 410 (Carl Zeiss, Inc.) confocal scanner.

Bottom Line: Proc.Natl.Real sparks differ from simulated ones mainly in having greater width.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Physiology, Rush University, Chicago, Illinois 60612, USA. erios@rush.edu

ABSTRACT
An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.

Show MeSH
Related in: MedlinePlus