Fast dual-excitation ratiometry with light-emitting diodes and high-speed liquid crystal shutters |
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Authors: | Fukano Takashi Shimozono Satoshi Miyawaki Atsushi |
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Institution: | Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan. |
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Abstract: | 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. |
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Keywords: | Fluorescence imaging Calcium imaging Cardiac cell |
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