SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data

Figures

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  Figure 1.. Demonstration of main features of SparkMaster 2 (SM2). A, Sample segmentation of a recording using SM2 (left) vs the original SparkMaster (right). B, An illustration of long spark detection by both SM2 and SparkMaster.

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  Figure 2.. SparkMaster 2 (SM2) controls. SM2 may be controlled either using a graphical user interface (A) or by writing Python scripts (B). The GUI provides access to setting of basic parameters (A1), advanced parameters changing details of analysis procedures (A2), single-recording analysis (A3), and other functions, including batch processing and parameter saving/loading (A4). The color overlays are added only for the purpose of this figure and are not present in the actual software.

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  Figure 3.. Using SparkMaster 2 (SM2) to analyze the effect of increasing [Ca]i on Ca release events. A, A comparison of the Ca release patterns in 5 distinct permeabilized cells exposed to a variety of calcium concentrations. B, A summary of frequencies of Ca sparks, miniwaves, and waves, in the 5 recordings above. C, A comparison of calcium spark decay time constants across the different concentrations (using standard boxplots in blue and the underlying data points in orange). To focus this analysis on sparks only, Ca miniwaves and waves were excluded, as well as sparks with decay time constant over 120 ms, indicating an incorrectly segmented object or the failure of the time constant fitting procedure.

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  Figure 4.. Comparing spark frequency following large release events vs other areas. A, An illustration of a box lasting 150 ms following a wave (blue) and a same-size box in a randomly selected position within the recording (orange). B, The difference.

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  Figure 5.. Comparison of SparkMaster 2 (SM2) and human annotators using synthetic data. A, An example of synthetic data produced, with a synthetic image to the left and a mask of ground truth spark segmentation to the right. B, The comparison of the performance of the 3 annotation systems. SparkMaster used thresholds of 3.4 and 3.8 for the more- and less-sensitive version respectively. SM2 used the thresholds of 0.45 and 0.6 for the more- and less-sensitive annotation (the threshold values are not comparable between SparkMaster and SM2). The total number of false negatives and false positives across 30 images in the dataset is shown.

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  Figure 6.. Application of SparkMaster 2 (SM2) to distinct cell types and conditions. A, Paced rabbit atrial cells loaded with Fluo-4. B, Mouse vascular smooth muscle cells imaged using a spinning disk confocal microscope and the Ca dye of Cal-520. Top image shows recorded images, with yellow line being used to extract the line-scan recording underneath. C and D, Analogous recordings of vascular smooth muscle cells, using Fluo-4 and GcaMP2, respectively to image the Ca sparks. Performance of the original SparkMaster on these recordings is shown and discussed in Supplemental Material S3.