Auto-luminescent genetically encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level

Kenta Saito; Kentaro Kobayashi; Takeharu Nagai

doi:10.1117/12.905065

23 December 2011 Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺ imaging at the single cell level

Kenta Saito, Kentaro Kobayashi, Takeharu Nagai

Author Affiliations +

Proceedings Volume 8204, Smart Nano-Micro Materials and Devices; 820403 (2011) https://doi.org/10.1117/12.905065
Event: SPIE Smart Nano + Micro Materials and Devices, 2011, Melbourne, Australia

Abstract

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. We have applied this strategy to develop an autoluminescent Ca²⁺ indicator, BRAC, which is composed of Ca²⁺-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. With this BRAC, we succeeded visualization of Ca²⁺ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca²⁺-independent signal drifts due to change in cell shape, focus shift, etc. Taking advantage of the bioluminescence imaging property that does not require external excitation light, BRAC might become a powerful tool applicable in conjunction with so-called optogenetic technology by which we can control cellular and protein function by light illumination.

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Kenta Saito, Kentaro Kobayashi, and Takeharu Nagai "Auto-luminescent genetically encoded ratiometric indicator for real-time Ca²⁺ imaging at the single cell level", Proc. SPIE 8204, Smart Nano-Micro Materials and Devices, 820403 (23 December 2011); https://doi.org/10.1117/12.905065

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