Automatic Detection and Classification of Ca²⁺ Release Events in Line- and Frame-Scan Images

Analysis of Ca²⁺ signals obtained in various cell types (i.e., cardiomyocytes) is always a tradeoff between acquisition speed and signal/noise ratio of the fluorescence signal. This becomes especially apparent during fast two- or three-dimensional confocal imaging when local intracellular fluorescence signals originating from Ca²⁺ release from intracellular Ca²⁺ stores (e.g., sarcoplasmic reticulum) need to be examined. Mathematical methods have been developed to remedy a high noise level by fitting each pixel with a transient function to "denoise" the image. So far, current available analytical approaches have been impaired by a number of constraints (e.g., inability to fit local, concurrent, and consecutive events) and the limited ability to customize implementation. Here, we suggest a, to our knowledge, novel approach for detailed analysis of subcellular micro-Ca²⁺ events based on pixel-by-pixel denoising of confocal frame- and line-scan images. The algorithm enables spatiotemporally overlapping events (e.g., a Ca²⁺ spark occurring during the decaying phase of a Ca²⁺ wave) to be extracted so that various types of Ca²⁺ events can be detected at a pixel time level of precision. The method allows a nonconstant baseline to be estimated for each pixel, foregoing the need to subtract fluorescence background or apply self-ratio methods before image analysis. Furthermore, by using a clustering algorithm, identified single-pixel events are grouped into "physiologically relevant" Ca²⁺ signaling events spanning multiple pixels (sparks, waves, puffs, transients, etc.), from which spatiotemporal event parameters (e.g., full duration at half maximal amplitude, full width at half maximal amplitude, amplitude, wave speed, rise, and decay times) can be easily extracted. The method was implemented with cross-platform open source software, providing a comprehensive and easy-to-use graphical user interface enabling rapid line-scan images and rapid frame-scan image sequences (up to 150 frames/s) to be analyzed and repetitive Ca²⁺ events (Ca²⁺ sparks and Ca²⁺ puffs) originating from clusters of Ca²⁺ release channels located in the sarcoplasmic reticulum membrane (ryanodine receptors and inositol 1,4,5-trisphosphate receptors) of isolated cardiomyocytes to be examined with a high level of precision.