Dual-excitation ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]). These dyes are excited alternately at two different wavelengths, and the pair of intensity measurements must be collected sequentially. Therefore, it is difficult to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples. Here, we present a novel but simple dual-excitation ratiometric method which overcomes this problem. By the use Of Our home-made illuminator, each sample is illuminated by two orthogonal linear polarized lights of different wavelengths. Fluorescence images are captured by two CCD cameras through two analyzers, whose polarization directions are at right angles. This methodology allows us to perform simultaneous measurements of any dual-excitation ratiometric dye, and we demonstrate its validity by monitoring [Ca2+] changes in rat cardiac muscle cells loaded with Fura Red. (C) 2004 Elsevier Inc. All rights reserved.