Question: 

Real-time imaging of fast signaling events has yielded important insights and has greatly fostered our understanding of fundamental processes such as subcellular calcium signaling in cardiac and neuronal cells. However, scanning technology has pushed the edge of acquisition speeds into dimensions in which the level of single pixel noise has become limiting for the interpretation of the data.

Methodology: 

Here we propose a robust analytical strategy to overcome such technical limitations in the imaging of calcium handling in excitable cells, such as cardiac myocytes, for which signaling (i.e. excitation-contraction coupling, ECC) occurs on the millisecond timescale. The analytical strategy compromises a pixel-wise fitting approach using high-speed 2-dimensional confocal calcium data and a mathematical approach designed to reconstruct local calcium transients.

Results: 

This approach not only produces virtually noise-free single pixel fluorescence data originating from subcellular volumes as small as 0.025 fl. It also allows extraction of 2D information on signaling events such as calcium induced calcium release in a very robust manner.

Conclusion: 

Such an analytical approach enabled us to tackle important questions of cardiac ECC under physiological conditions but also helped to reveal novel information about the pro-arrhythmogenic calcium alternans by demonstrating that microscopic alternans preceeds macroscopic alternans. The applicability of such an analytical, pixel-wise fitting approach can be transferred to other biological systems and may thus help to overcome present technical limitations such as diminished signal to noise levels in high-speed live cell imaging.

This work was supported by the DFG, BMBF and the Medical Faculty.