Comparing synthetic refocusing to deconvolution for the extraction of neuronal calcium transients from light fields.

Significance: Light-field microscopy (LFM) enables fast, light-efficient, volumetric imaging of neuronal activity with calcium indicators. Calcium transients differ in temporal signal-to-noise ratio (tSNR) and spatial confinement when extracted from volumes reconstructed by different algorithms. Aim: We evaluated the capabilities and limitations of two light-field reconstruction algorithms for calcium fluorescence imaging. Approach: We acquired light-field image series from neurons either bulk-labeled or filled intracellularly with the red-emitting calcium dye CaSiR-1 in acute mouse brain slices. We compared the tSNR and spatial confinement of calcium signals extracted from volumes reconstructed with synthetic refocusing and Richardson-Lucy three-dimensional deconvolution with and without total variation regularization. Results: Both synthetic refocusing and Richardson-Lucy deconvolution resolved calcium signals from single cells and neuronal dendrites in three dimensions. Increasing deconvolution iteration number improved spatial confinement but reduced tSNR compared with synthetic refocusing. Volumetric light-field imaging did not decrease calcium signal tSNR compared with interleaved, widefield image series acquired in matched planes. Conclusions: LFM enables high-volume rate, volumetric imaging of calcium transients in single cell somata (bulk-labeled) and dendrites (intracellularly loaded). The trade-offs identified for tSNR, spatial confinement, and computational cost indicate which of synthetic refocusing or deconvolution can better realize the scientific requirements of future LFM calcium imaging applications.