Apparent calcium spark properties and fast-scanning 2D confocal imaging modalities

[Display omitted] •Fast 2D over time confocal microscopes enable high-content acquisition of cardiac Ca2+ sparks.•Data from point scanners suffer from low pixel photon fluxes and low signal-to-noise ratios.•Data from 2D-array scanners provide high pixel photon fluxes and 2-3 fold increased signal-to-noise ratios.•In thick samples, 2D-array scanners suffer from inter-pinhole crosstalk.•Apparent spark properties such as frequency, amplitude, decay time and spatial spread are dependent on the particular imaging modality. Ca2+ sparks are instrumental to understand physiological and pathological Ca2+ signaling in the heart. High-speed two spatially dimensional (2D) confocal imaging (>120 Hz) enables acquisition of sparks with high-content information, however, owing to a wide variety of different acquisition modalities the question arises: how much they reflect the “true” Ca2+ spark properties. To address this issue, we compared a fast point and a 2D-array scanner equipped with a range of different detectors. As a quasi-standard biological sample, we employed Ca2+ sparks in permeabilized and intact mouse ventricular myocytes and utilized an unbiased, automatic Ca2+ spark analysis tool, iSpark. Data from the point scanner suffered from low pixel photon fluxes (PPF) concomitant with high Poissonian noise. Images from the 2D-array scanner displayed substantially increased PPF, lower Poissonian noise and almost 3-fold increased sign-to-noise ratios. Noteworthy, data from the 2D scanner suffered from considerable inter-pinhole crosstalk evident for the permeabilized cells. Spark properties, such as frequency, amplitude, decay time and spatial spread were distinctly different for any scanner/detector combination. Our study reveals that the apparent Ca2+ spark properties differ dependent on the particular recording modality and set-up employed, quantitatively.