3D high-speed multifocal multiphoton microscopy in muscle and wavelet analysis of fast elementary calcium release events

Recent approaches to record and analyze skeletal muscle elementary calcium release events (ECRE) in three dimensions (XYT) were restricted to short time series and small areas of interest. Here, we present a combined method for XYT analysis of ECRE using high-speed multifocal multiphoton calcium imaging and a wavelet based automated denoising and detection algorithm. Images were analyzed with the 'a trous' implementation of the wavelet transform. 'Hard-threshold' wavelet denoising resulted in markedly improved signal-to-noise ratios compared to low-pass filtering. Automated detection of ECRE was performed across wavelet scales and single events were tracked within an image sequence using 3D segmentation. With this combined approach, the morphological dynamics of fast ECRE in mammalian skeletal muscle fibres could be faithfully monitored with high spatial and temporal resolution. Our method may provide an improved tool for the facilitated detection of ECRE in muscle in three dimensions even under conditions of undisturbed calcium homeostasis.