New statistical methods enhance imaging of cameleon fluorescence resonance energy transfer in cultured zebrafish spinal neurons.

Cameleons are genetically encoded fluorescence resonance energy transfer (FRET)-based Ca(2+) indicators. Attempts to use cameleons to detect neural activity in vertebrate systems have been largely frustrated by the small FRET signal, in contradistinction to the higher signals seen in Drosophila and Caenorhabditis elegans. We have developed a statistical optimization method capable of detecting small ratiometric signals in noisy imaging data, called statistical optimization for the analysis of ratiometric signals. Using this method, we can detect and estimate anticorrelated ratiometric signals with subcellular resolution in cultured, dissociated zebrafish spinal neurons expressing cameleon or loaded with fluo-4 and fura-red. This method may make it possible to use yellow cameleons for measuring neural activity at high resolution in transgenic animals.