von Hofe Julian, Abacousnac Jatin, Chen Mechi, Sasazawa Moeka, Javér Kristiansen Ida, Westrey Soren, Grier David G, Saurabh Saumya
Department of Chemistry, New York University, New York, New York 10003, United States.
Department of Physics and Center for Soft Matter Research, New York University, New York, New York 10003, United States.
J Am Chem Soc. 2025 Jul 23;147(29):25242-25253. doi: 10.1021/jacs.5c02947. Epub 2025 Jul 8.
Biomolecular condensates are essential for cellular organization and function, yet understanding how chemical and physical factors govern their formation and dynamics has been limited by a lack of noninvasive measurement techniques. Conventional microscopy methods often rely on fluorescent labeling and substrate immobilization, which can perturb the intrinsic properties of condensates. To overcome these challenges, we apply label-free, contact-free holographic video microscopy to study the behavior of a condensate-forming protein in vitro. This technique enables rapid, high-throughput, and precise measurements of individual condensate diameters and refractive indexes, providing unprecedented insight into size distributions and dense-phase macromolecular concentrations over time. Using this method, we investigate the kinetics of droplet growth, aging, and equilibrium dynamics in the model condensate-forming protein PopZ. By systematically varying the concentration and valence of cations, we uncover how multivalent ions influence condensate organization and dynamics, a hypothesis we further test using super-resolution microscopy. Our findings reveal that PopZ droplet growth deviates from classical models such as Smoluchowski coalescence and Ostwald ripening. Instead, we show that condensate growth is consistent with gelation at the critical overlap concentration. Holographic microscopy offers significant advantages over traditional techniques, such as differential interference contrast microscopy, delivering reproducible measurements and capturing condensate dynamics with unparalleled precision. This work highlights the power of holographic microscopy to probe the material properties and mechanistic underpinnings of biomolecular condensates, paving the way for deeper insights into their roles in synthetic systems.
生物分子凝聚物对于细胞组织和功能至关重要，然而，由于缺乏非侵入性测量技术，化学和物理因素如何控制其形成和动力学的理解一直受到限制。传统的显微镜方法通常依赖于荧光标记和底物固定，这可能会干扰凝聚物的固有特性。为了克服这些挑战，我们应用无标记、非接触式全息视频显微镜来研究体外形成凝聚物的蛋白质的行为。该技术能够对单个凝聚物的直径和折射率进行快速、高通量和精确的测量，为随时间变化的尺寸分布和密集相大分子浓度提供了前所未有的见解。使用这种方法，我们研究了模型凝聚物形成蛋白PopZ中液滴生长、老化和平衡动力学的过程。通过系统地改变阳离子的浓度和价态，我们揭示了多价离子如何影响凝聚物的组织和动力学，我们使用超分辨率显微镜进一步验证了这一假设。我们的研究结果表明，PopZ液滴的生长偏离了经典模型，如斯莫卢霍夫斯基聚结和奥斯特瓦尔德熟化。相反，我们表明凝聚物的生长与临界重叠浓度下的凝胶化一致。全息显微镜相对于传统技术，如微分干涉对比显微镜，具有显著优势，能够提供可重复的测量结果，并以前所未有的精度捕捉凝聚物的动力学。这项工作突出了全息显微镜在探测生物分子凝聚物的材料特性和机制基础方面的强大能力，为更深入了解它们在合成系统中的作用铺平了道路。
