Abstract
Previous studies have used analysis of Ca2+ sparks extensively to investigate both normal and pathological Ca2+ regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca2+ sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca2+ spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca2+ sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.
Highlights
Ca2þ sparks are elementary, localized Ca2þ release events that result from the activation of ryanodine receptor (RyR) clusters within the sarcoplasmic reticulum (SR) of cardiac, skeletal, or smooth muscle [1]
This study describes the development and characterization of software to enable automatic detection and analysis of Ca2þ sparks in x-y image stacks, implemented as a plugin for ImageJ. xySpark provides an interactive graphical user interface and includes novel methods to enable accurate identification of cells within confocal fluorescence images, compensation for slow changes in background fluorescence during data collection, and options that allow exclusion of aberrant events based on spatial characteristics
Our aim in this study was to develop an algorithm to automatically detect and analyze Ca2þ sparks in x-y confocal image stacks obtained from quiescent myocytes, i.e., where the background cell fluorescence is relatively constant and spontaneous or triggered global Ca2þ transients are absent
Summary
Ca2þ sparks are elementary, localized Ca2þ release events that result from the activation of ryanodine receptor (RyR) clusters within the sarcoplasmic reticulum (SR) of cardiac, skeletal, or smooth muscle [1]. The great majority of past studies on Ca2þ sparks have involved laser scanning confocal microscopy (LSCM) operating in line-scan mode, where the data are collected from a single line of pixels. In part, this reflects the fact that one can obtain adequate temporal resolution using relatively lowspecification confocal systems. The development of software to automate detection of Ca2þ sparks in linescan images markedly accelerated and simplified the process of Ca2þ spark detection and analysis [13]. Its subsequent implementation as a plugin within the open-source image analysis environment ImageJ was widely adopted [14], providing the additional benefit that data collected by different labs could be compared more readily
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