Multidimensional detection and analysis of Ca2+ sparks in cardiac myocytes

Abstract

Examining calcium spark morphology and its relationship to the structure of the cardiac myocyte offers a direct means of understanding excitation-contraction coupling mechanisms. Traditional confocal line scanning achieves excellent temporal spark resolution but at the cost of spatial information in the perpendicular dimension. To address this, we developed a methodology to identify and analyze sparks obtained via two-dimensional confocal or charge-coupled device microscopy. The technique consists of nonlinearly subtracting the background fluorescence, thresholding the data on the basis of noise level, and then localizing the spark peaks via a generalized extrema test, while taking care to detect and separate adjacent peaks. In this article, we describe the algorithm, compare its performance to a previously validated spark detection algorithm, and demonstrate it by applying it to both a synthetic replica and an experimental preparation of a two-dimensional isotropic myocyte monolayer exhibiting sparks during a calcium transient. We find that our multidimensional algorithm provides better sensitivity than the conventional method under conditions of temporally heterogeneous background fluorescence, and the inclusion of peak segmentation reduces false negative rates when spark density is high. Our algorithm is robust and can be effectively used with different imaging modalities and allows spark identification and quantification in subcellular, cellular, and tissue preparations.

FIGURE 1

Example of raw fluorescence data obtained by a CCD camera from an engineered two-dimensional cardiac tissue. (A–C) A single (x,y) fluorescence (F) map taken at t = 2.38 s labeled with (A) di-8-ANEPPS, highlighting the cell membranes, (B) DAPI, highlighting the nuclei, and (C) fluo-4, showing [Ca²⁺]_i. (D,E) Development in time of F along the dotted lines labeled D and E shown in panel C. The red boxes outline the same spark in panels D and E. The periodic pattern represents spontaneous, large-scale Ca²⁺ transients that occupy the entire tissue area simultaneously. (F) Temporal trace at the location shown in panel C with a white box, illustrating the spontaneous Ca²⁺ transients and the spark highlighted in panels D and E. Spatial scale bar is 20 μm, temporal scale bar is 0.5 s. A movie of the Ca²⁺ fluorescence shown in panel C is provided as Supplementary Material.

FIGURE 2

Example of adjacent spark segmentation. (A) Two neighboring sparks, highlighted as three regions in (x,y,t) space: suprathreshold regions (green), relative maxima (blue), actual maxima (black dots). A plane intersecting the actual maxima and bisecting the relative maxima and suprathreshold regions is shown in black outline. (B) The Euclidean distance transformation on the plane shown in panel A with respect to the relative maxima. The actual maxima are shown as black dots. The watershed line is shown in red, dividing the space into two regions, labeled 1 and 2. (C) The same plot as that in panel A, with the watershed surface superimposed in red, dividing the (x,y,t) space into two regions, labeled 1 and 2.

FIGURE 3

Creation of synthetic sparks. (A) A sample frame from t = 1 s. Scale bar is 20 μm. (B,C) Space-time plot of ΔF/F₀ along the line shown in panel A, showing synthetic sparks in the presence of (B) added baseline fluorescence noise and (C) added baseline fluorescence noise in addition to the fluorescence change associated with tissue-wide Ca²⁺ reuptake.

FIGURE 4

(A) Sensitivity of the algorithm as a function of synthetic spark normalized amplitude for various threshold criteria, Cri. (B) False negative rate (blue) and positive predictive values (green) as a function of synthetic spark normalized amplitude for values of Cri. The average results of eye detection by a coauthor were fitted to a sigmoid curve and given as the solid red lines in panels A and B.

FIGURE 5

(A) Accuracy of fullwidth, half-maximum (FWHM) estimation in the x-direction using the detection algorithm on DS0 (noise added, •) and DS2 (no noise, ○) synthetic data. All synthetic sparks have the same FWHM but varying amplitudes, given by the horizontal black dotted line. Error bars show SEM. (B) Histograms of the estimated FWHM measurements in the x-direction from the DS0 synthetic data sets, given as the average number of detected events per image. The actual FWHM in x is given by the vertical black dotted line.

FIGURE 6

Statistical parameters obtained for the conventional algorithm for uniform baseline fluorescence (top row) and Ca²⁺ reuptake (bottom row). (A and C) Sensitivities as a function of synthetic spark normalized amplitude for various threshold criteria, Cri. (B and D) False negative rate (blue) and positive predictive values (green) as a function of synthetic spark normalized amplitude for values of Cri. The average results of eye detection by a coauthor were fitted to a sigmoid curve and given as the solid red lines. Curves shifted to the right indicate diminished detection properties. Note the change in x axis limits between panels A and B and panels C and D.

FIGURE 7

Number of false negatives as a function of spark density for synthetic data. Circles and fitted solid line indicate those detected using the conventional algorithm; plus signs and fitted dotted line indicate those detected using the multidimensional algorithm. Only those sparks with ΔF/F₀ > 0.3 are considered.

FIGURE 8

(A) Estimated FWHM values in x and y as a function of estimated amplitude using the detection algorithm on data collected from Ca²⁺ transient events (n = 21). Error bars show mean ± SE. (B) Histograms of the estimated FWHM values in x and y for the experimental data sets.

FIGURE 9

(A) An example of spark detection algorithm performance of experimental data taken at t = 89 ms; t = 0 is the point at which the tissue fluorescence has fallen to 25% of its maximum intensity. (Left) [Ca²⁺]_i fluorescence of the tissue. Red rectangles indicate the boundaries of a detected spark in (x,y) and borders of nuclei are shown in yellow outline. Scale bar is 20 μm. (Right) (x,y,t) image sequence (x,y, horizontal axes; t, vertical axis; t = 0 at the bottom face) with the red boxes indicating the (x,y,t) boundaries of a detected spark. The image frame shown in the left panel is illustrated as a horizontal cross section in the right panel. (B) Spatial map of spark frequency. Detected spark locations are expressed as a 2-D smoothed histogram with 25 × 25 μm² bins. Scale bar is 20 μm. Borders of nuclei shown with a white outline. (C) Image of ΔF/F₀ smoothed with 3 × 3 spatial average filter depicting the development of a perinuclear spark. Black outline indicates contour of nucleus labeled with an asterisk in panel B. Scale bar is 5 μm.