Selective Plane Illumination Microscopy (SPIM) is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on F?rster Resonance Energy Transfer (FRET). Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+) probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+) dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+) signal percolation, predicted in previous studies, has been directly visualized.
Arabidopsis Calcium