Imaging Changes in Cytoplasmic Calcium Using the Yellow Cameleon 3.6 Biosensor and Confocal Microscopy

Changes in the concentration of cytoplasmic calcium, [Ca²⁺ ]_cyt are central regulators in many cellular signal transduction pathways including many lipid-mediated regulatory networks. Given this central role that [Ca²⁺ ] has during plant growth, monitoring spatial and temporal [Ca²⁺ ] dynamics can reveal a critical component of cellular physiology. Here, we describe the measurement of [Ca²⁺ ]_cyt in Arabidopsis root cells using plants expressing Yellow Cameleon 3.6 (YC 3.6). YC3.6 is a Ca²⁺ -sensitive biosensor where the intensity of its fluorescence resonance energy transfer (FRET) signal changes as the Ca²⁺ level within the cell rises and falls. The FRET from this calcium reporter can be visualized using confocal microscopy and the resultant images converted to a quantitative map of the levels of Ca²⁺ using an approach called ratio analysis.