Fast dual-excitation ratiometry with light-emitting diodes and high-speed liquid crystal shutters

Dual-excitation ratiometric dyes permit quantitative measurements of Ca2+ concentrations ([Ca2+]s), by minimizing the effects of several artifacts that are unrelated to changes in [Ca2+]. These dyes are excited at two different wavelengths, and the resultant fluorescence intensities are measured sequentially. Therefore, it is difficult to follow fast [Ca2+] dynamics or [Ca2+] changes in highly motile cell samples. To overcome this problem, we have developed a new dual-excitation ratiometry system that employs two high-power light-emitting diodes (LEDs), two high-speed liquid crystal shutters, and a CCD camera. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10–100 Hz depending on the signal intensity. We demonstrate the effectiveness of this system by monitoring changes in [Ca2+] in cardiac muscle cells loaded with Fura-2.