XVI International Botanical Congess

Yellow cameleon -2 (YC2, see Nature 388:882) is a GFP-based reporter which increases FRET with increasing (Ca2+). It was expressed in onion and By-2 tobacco cells for high-resolution visualization of spatiotemporal distributions of Ca2+ by means of deconvolution microscopy. Calibration was accomplished by buffering YC2 protein. With 2 CCD cameras in simultaneous use, about 20 256 x 256 x 64 images (with 0.25 x 0.25 x 0.25 ?m voxels) can be collected in a time-course of a cell in which cytoplasmic streaming is slow. Accuracy of deconvolved images is far greater than for 2D collections, thought the latter permit time-courses with more time points and during fast streaming. At a detailed level, Ca2+ appears markedly nonhomogeneous even in non-stimulated cells, however, allowance must be made for some pH sensitivity of FRET by YC2. Time-courses following application of asymmetric mechanical pressure and of hypoosmotic medium permit comparison of spatial, temporal, and intensity sensitivity of this method versus others.