Kim Sang-Yeob, Arai Yoshiyuki, Tani Tomomi, Takatsuka Hirofumi, Saito Yoshiharu, Kawashima Takayuki, Kawakami Shojiro, Miyawaki Atsushi, Nagai Takeharu
Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.
The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
Microscopy (Oxf). 2017 Apr 1;66(2):110-119. doi: 10.1093/jmicro/dfw110.
Förster resonance energy transfer (FRET) has been widely used to design indicators for biomolecules. Conventional FRET-based indicators enable quantitative measurements of analyzes by calculating the ratio between donor and acceptor fluorophores. However, such 'hetero-FRET'-based indicators, which use multiple differently colored fluorophores, restrict the simultaneous use of other colors of fluorescent molecules. To overcome this problem, we developed a 'homo-FRET'-based Ca2+ indicator, W-Cameleon, composed of two identical yellow fluorescent proteins. The binding of Ca2+ to the indicator induces a change in FRET efficiency, which in turn transforms into changes in fluorescence anisotropy. Given that the fluorescence polarization is depolarized by light passing through a high numerical aperture lens and reflecting on a dichroic mirror, we also developed a microscopy technique that reliably detects fluorescence anisotropy with high precision. Our design is aided by photonic-crystal technology, to compensate for the fluorescence depolarization. We thereby succeeded in the simultaneous visualization of three individual intracellular events by using three different fluorescent indicators. Our system may contribute to an expansion of the number of events that can be observed, which will enable a more quantitative understanding of biological phenomena.
荧光共振能量转移（FRET）已被广泛用于设计生物分子指示剂。传统的基于FRET的指示剂通过计算供体和受体荧光团之间的比率来实现分析物的定量测量。然而，这种基于“异质FRET”的指示剂使用多种不同颜色的荧光团，限制了其他颜色荧光分子的同时使用。为了克服这个问题，我们开发了一种基于“同质FRET”的Ca2+指示剂W-Cameleon，它由两个相同的黄色荧光蛋白组成。Ca2+与指示剂的结合会引起FRET效率的变化，进而转化为荧光各向异性的变化。鉴于荧光偏振会被穿过高数值孔径透镜并在二向色镜上反射的光去极化，我们还开发了一种能够高精度可靠检测荧光各向异性的显微镜技术。我们的设计借助了光子晶体技术来补偿荧光去极化。通过使用三种不同的荧光指示剂，我们成功地同时可视化了三个独立的细胞内事件。我们的系统可能有助于增加可观察事件的数量，从而能够更定量地理解生物现象。
