TY - JOUR
T1 - Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level
AU - Saito, Kenta
AU - Hatsugai, Noriyuki
AU - Horikawa, Kazuki
AU - Kobayashi, Kentaro
AU - Matsu-Ura, Toru
AU - Mikoshiba, Katsuhiko
AU - Nagai, Takeharu
PY - 2010
Y1 - 2010
N2 - Background: 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. Methodology/Principal Findings: We applied BRET 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. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful 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. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2++ imaging not only in single live cells but also in small living subjects.
AB - Background: 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. Methodology/Principal Findings: We applied BRET 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. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful 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. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2++ imaging not only in single live cells but also in small living subjects.
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U2 - 10.1371/journal.pone.0009935
DO - 10.1371/journal.pone.0009935
M3 - Article
C2 - 20376337
AN - SCOPUS:77955947906
SN - 1932-6203
VL - 5
JO - PloS one
JF - PloS one
IS - 4
M1 - e9935
ER -