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Sci. STKE, 26 March 2002
Vol. 2002, Issue 125, p. pl4
[DOI: 10.1126/stke.2002.125.pl4]

PROTOCOLS

Confocal Imaging of Subcellular Ca2+ Concentrations Using a Dual-Excitation Ratiometric Indicator Based on Green Fluorescent Protein

Satoshi Shimozono1,2, Takashi Fukano1, Takeharu Nagai1,3, Yutaka Kirino2, Hideaki Mizuno1, and Atsushi Miyawaki1*

1Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
2Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
3Structure and Function of Biomolecules, PRESTO, JST, Nittochi 535 Akinono-cho Nakagyo-ku Kyoto, 604-0847, Japan.

Abstract: Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca2+ indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca2+ imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca2+ using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca2+ waves from the cytosol to the nucleus and changes in Ca2+ concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.

*Corresponding author. Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan. Telephone, +81-48-467-5917; fax, +81-48-467-5924; e-mail, matsushi@brain.riken.go.jp.

Citation: S. Shimozono, T. Fukano, T. Nagai, Y. Kirino, H. Mizuno, A. Miyawaki, Confocal Imaging of Subcellular Ca2+ Concentrations Using a Dual-Excitation Ratiometric Indicator Based on Green Fluorescent Protein. Sci. STKE 2002, pl4 (2002).

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