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.
Fukano
Takashi
T
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.
Shimozono
Satoshi
S
Miyawaki
Atsushi
A
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D016257:Fura-2 / Q000008:administration & dosage*; D007089:Image Enhancement / Q000295:instrumentation* / Q000379:methods; D008029:Lighting; D050866:Liquid Crystals; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012666:Semiconductors; D012815:Signal Processing, Computer-Assisted
D002118:Calcium; D016257:Fura-2