11 February 2011 Development of ultra-sensitive Ca2+ indicators, yellow cameleon-nanos
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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.
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Takeharu Nagai, Takeharu Nagai, Kazuki Horikawa, Kazuki Horikawa, Tomoki Matsuda, Tomoki Matsuda, Kentaro Kobayashi, Kentaro Kobayashi, } "Development of ultra-sensitive Ca2+ indicators, yellow cameleon-nanos", Proc. SPIE 7910, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III, 79100G (11 February 2011); doi: 10.1117/12.871202; https://doi.org/10.1117/12.871202


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