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Proceedings Paper

Development of ultra-sensitive Ca2+ indicators, yellow cameleon-nanos
Author(s): Takeharu Nagai; Kazuki Horikawa; Tomoki Matsuda; Kentaro Kobayashi
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Paper Abstract

There are so many Ca2+ indicators such as Oregon green 488 BAPTA-1 and cameleon, all of which have moderate Ca2+ affinities (Kd > 150 nM). These indicators successfully report changing in Ca2+ concentration in most of cells. However, it is known that some cells are estimated to have a very low resting [Ca2+] and display very small ▵[Ca2+]s, which is below the detection limit of the indicators. To develop high-affinity indicators, we modified a FRET-based Ca2+ indicator, yellow cameleon (YC) 2.60 (Kd = 100 nM) because of the brightness and the large dynamic range. By engineering the Ca2+ sensing domain, we obtained a series of ultra-sensitive Ca2+ indicators. Their high Ca2+ affinities (Kd = 15, 30, and 50 nM) enabled detection of subtle Ca2+ transients associated with spontaneous network activity in zebrafish. Our measurements revealed that both the resting Ca2+ level and the amplitude of Ca2+ transients significantly differed by cell type and stimulation, indicating that the selection of indicators with the appropriate Kd is essential for successful in vivo Ca2+ imaging. A lineup of such indicators with finely tuned Kd values optimized to detect [Ca2+] from 10 nM to 100 nM would enable precise and reliable Ca2+ imaging.

Paper Details

Date Published: 12 February 2011
PDF: 7 pages
Proc. SPIE 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III, 79100G (12 February 2011); doi: 10.1117/12.871202
Show Author Affiliations
Takeharu Nagai, Hokkaido Univ. (Japan)
Japan Science and Technology Agency (Japan)
Kazuki Horikawa, Hokkaido Univ. (Japan)
Japan Science and Technology Agency (Japan)
Tomoki Matsuda, Hokkaido Univ. (Japan)
Kentaro Kobayashi, Hokkaido Univ. (Japan)


Published in SPIE Proceedings Vol. 7910:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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