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<p>Liquid–liquid phase separation leads to the formation of liquid droplets (LqDs) such as P granules in <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). 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&#xa0;mg/mL. In vivo measurements, we performed label-free imaging of <i>C. elegans.</i> Despite efforts to detect P granules within P lineage cells in both wild-type <i>C. elegans</i> 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.</p> Graphical abstract <p></p>

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Exploring liquid–liquid phase separation in vitro and in vivo using multimodal nonlinear optical imaging

  • 期刊论文

  • 发表日期：

    2025年3月20日

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  • Yusuke Murakami,
  • Mia Obuchi,
  • Hiroshi Kamizawa,
  • Shinichi Miyazaki,
  • Akihiro Kishimura,
  • Ryosuke Oketani,
  • Kotaro Hiramatsu,
  • Philippe Leproux,
  • Yu Hayashi,
  • Kentaro Shiraki,
  • Hideaki Kano

摘要

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.

Graphical abstract