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Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

Kong CH, Laver DR, Cannell MB - PLoS Comput. Biol. (2013)

Bottom Line: While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level.This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms.By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT
The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

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The effect of live myocytes on the confocal PSF.Examples of PSFs measured using (A) water and (B) oil immersion objectives on the coverslip (i) and on top of a live cardiac myocyte (ii) bathed in 1 mM Ca-Tyrode's solution are shown (see inset). Scale bars indicate 0.5 µm. (C) The intensity profiles across the peak intensity in (i) y and (ii) z are shown. The effect of the refractive index mismatch across the myocyte was more pronounced when using an oil immersion objective.
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pcbi-1002931-g002: The effect of live myocytes on the confocal PSF.Examples of PSFs measured using (A) water and (B) oil immersion objectives on the coverslip (i) and on top of a live cardiac myocyte (ii) bathed in 1 mM Ca-Tyrode's solution are shown (see inset). Scale bars indicate 0.5 µm. (C) The intensity profiles across the peak intensity in (i) y and (ii) z are shown. The effect of the refractive index mismatch across the myocyte was more pronounced when using an oil immersion objective.

Mentions: Images of yellow-green beads located on the coverslip (Fig. 2i) of a perfusion chamber and on top of live myocytes (Fig. 2ii) for both water- (Fig. 2A) and oil- (Fig. 2B) immersion objectives are shown. It is notable the recorded PSF is clearly distorted along the optical axis, an effect which we attribute to refractive index mismatch(es). A summary of PSF dimensions is given in Table 2, and it is clear that the distortion in z is most pronounced when using the oil-immersion objective (as might be expected), where the FWHM of the PSF was almost doubled. It is clear from these data that assumption of an idealized (i.e. diffraction limited) PSF during Ca spark and Ca blink recording would be erroneous.


Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

Kong CH, Laver DR, Cannell MB - PLoS Comput. Biol. (2013)

The effect of live myocytes on the confocal PSF.Examples of PSFs measured using (A) water and (B) oil immersion objectives on the coverslip (i) and on top of a live cardiac myocyte (ii) bathed in 1 mM Ca-Tyrode's solution are shown (see inset). Scale bars indicate 0.5 µm. (C) The intensity profiles across the peak intensity in (i) y and (ii) z are shown. The effect of the refractive index mismatch across the myocyte was more pronounced when using an oil immersion objective.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585382&req=5

pcbi-1002931-g002: The effect of live myocytes on the confocal PSF.Examples of PSFs measured using (A) water and (B) oil immersion objectives on the coverslip (i) and on top of a live cardiac myocyte (ii) bathed in 1 mM Ca-Tyrode's solution are shown (see inset). Scale bars indicate 0.5 µm. (C) The intensity profiles across the peak intensity in (i) y and (ii) z are shown. The effect of the refractive index mismatch across the myocyte was more pronounced when using an oil immersion objective.
Mentions: Images of yellow-green beads located on the coverslip (Fig. 2i) of a perfusion chamber and on top of live myocytes (Fig. 2ii) for both water- (Fig. 2A) and oil- (Fig. 2B) immersion objectives are shown. It is notable the recorded PSF is clearly distorted along the optical axis, an effect which we attribute to refractive index mismatch(es). A summary of PSF dimensions is given in Table 2, and it is clear that the distortion in z is most pronounced when using the oil-immersion objective (as might be expected), where the FWHM of the PSF was almost doubled. It is clear from these data that assumption of an idealized (i.e. diffraction limited) PSF during Ca spark and Ca blink recording would be erroneous.

Bottom Line: While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level.This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms.By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT
The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

Show MeSH
Related in: MedlinePlus