Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Methods Mol Biol. 2021;2274:127-138. doi: 10.1007/978-1-0716-1258-3_12.
Spectral overlaps in fluorescence (FL) and bioluminescence (BL) commonly cause optical cross talks. The present protocol introduces five different lineages of coelenterazine (CTZ) analogues, which have selectivity to a specific luciferase, and thus cross talk-free. For example, some CTZ analogues with ethynyl or styryl groups display dramatically biased BL to specific luciferases and pH by modifying the functional groups at the C-2 and C-6 positions of the imidazopyridinne backbone of CTZ. The optical cross talk-free feature is exemplified with the multiplex system, which simultaneously illuminated antiestrogenic and rapamycin activities without optical cross talks. This unique protocol contributes to specific and high-throughput BL imaging of multiple optical readouts in mammalian cells without optical contamination.
荧光（FL）和生物发光（BL）的光谱重叠通常会导致光学串扰。本方案介绍了 5 种不同谱系的腔肠素（CTZ）类似物，它们对特定的荧光素有选择性，因此没有光学串扰。例如，一些带有乙炔基或苯乙烯基的 CTZ 类似物通过修饰 CTZ 咪唑并吡啶骨架的 C-2 和 C-6 位的官能团，对特定的荧光素和 pH 值表现出明显的 BL 偏置。光学无串扰的特点在多重系统中得到了例证，该系统可以同时对雌激素拮抗剂和雷帕霉素的活性进行照明，而不会产生光学串扰。该独特方案有助于在哺乳动物细胞中进行多个光学读出的特异性和高通量 BL 成像，而不会产生光学污染。
