Murakami Yusuke, Obuchi Mia, Kamizawa Hiroshi, Miyazaki Shinichi, Kishimura Akihiro, Oketani Ryosuke, Hiramatsu Kotaro, Leproux Philippe, Hayashi Yu, Shiraki Kentaro, Kano Hideaki
Ph. D. Program in Humanics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577, Japan.
International Institute for Integrative Sleep Medicine (WPI-IIIS), 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.
Anal Sci. 2025 Mar 20. doi: 10.1007/s44211-025-00747-3.
Liquid-liquid phase separation leads to the formation of liquid droplets (LqDs) such as P granules in Caenorhabditis elegans (C. elegans). In this study, we demonstrate the label-free visualization of LqDs using multimodal nonlinear optical imaging both in vitro and in vivo. In vitro measurements with polymerized adenine [poly(A)], we found significantly higher poly(A) concentrations in LqDs compared to surrounding solutions, with the limit of detection (LoD) of 32 mg/mL. In vivo measurements, we performed label-free imaging of C. elegans. Despite efforts to detect P granules within P lineage cells in both wild-type C. elegans and green fluorescent protein (GFP)-tagged strains, no clear RNA-specific signals were observed. This indicates that the RNA concentration in P granules is lower than anticipated and falls below our in vitro LoD. These results underscore the challenges of label-free RNA detection in P granules.
液-液相分离导致液滴（LqDs）的形成，如秀丽隐杆线虫（C. elegans）中的P颗粒。在本研究中，我们展示了使用多模态非线性光学成像在体外和体内对LqDs进行无标记可视化。在用聚合腺嘌呤[poly(A)]进行的体外测量中，我们发现LqDs中的poly(A)浓度显著高于周围溶液，检测限（LoD）为32 mg/mL。在体内测量中，我们对秀丽隐杆线虫进行了无标记成像。尽管努力在野生型秀丽隐杆线虫和绿色荧光蛋白（GFP）标记菌株的P谱系细胞内检测P颗粒，但未观察到明确的RNA特异性信号。这表明P颗粒中的RNA浓度低于预期，且低于我们的体外LoD。这些结果强调了在P颗粒中进行无标记RNA检测的挑战。
