Most chemistry and biology occurs in solution, in which conformational dynamics and complexation underlie behaviour and function. Single-molecule techniques¹ are uniquely suited to resolving molecular diversity and new label-free approaches are reshaping the power of single-molecule measurements. A label-free single-molecule method^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16} capable of revealing details of molecular conformation in solution^17,18 would allow a new microscopic perspective of unprecedented detail. Here we use the enhanced light–molecule interactions in high-finesse fibre-based Fabry–Pérot microcavities^19,20,21 to detect individual biomolecules as small as 1.2 kDa, a ten-amino-acid peptide, with signal-to-noise ratios (SNRs) >100, even as the molecules are unlabelled and freely diffusing in solution. Our method delivers 2D intensity and temporal profiles, enabling the distinction of subpopulations in mixed samples. Notably, we observe a linear relationship between passage time and molecular radius, unlocking the potential to gather crucial information about diffusion and solution-phase conformation. Furthermore, mixtures of biomolecule isomers of the same molecular weight and composition but different conformation can also be resolved. Detection is based on the creation of a new molecular velocity filter window and a dynamic thermal priming mechanism that make use of the interplay between optical and thermal dynamics^22,23 and Pound–Drever–Hall (PDH) cavity locking²⁴ to reveal molecular motion even while suppressing environmental noise. New in vitro ways of revealing molecular conformation, diversity and dynamics can find broad potential for applications in the life and chemical sciences.

大多数化学和生物学发生在溶液中，其中构象动力学和络合是行为和功能的基础。单分子技术¹特别适合解决分子多样性问题，新的无标记方法正在重塑单分子测量的能力。无标记单分子方法^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16}能够揭示溶液^17,18中分子构象的细节提供前所未有的细节的新微观视角。在这里，我们利用基于高精细纤维的法布里-珀罗微腔中增强的光-分子相互作用^19,20,21来检测小至 1.2 kDa 的单个生物分子（一种十氨基酸肽），具有信噪比(SNR) >100，即使分子未标记并在溶液中自由扩散。我们的方法提供 2D 强度和时间剖面，从而能够区分混合样本中的亚群。值得注意的是，我们观察到通过时间和分子半径之间的线性关系，释放了收集有关扩散和溶液相构象的关键信息的潜力。此外，还可以解析具有相同分子量和组成但不同构象的生物分子异构体的混合物。检测基于新的分子速度过滤窗口和动态热启动机制的创建，该机制利用光学和热动力学^22,23以及 Pound-Drever-Hall (PDH) 腔锁定²⁴之间的相互作用来揭示分子运动，甚至同时抑制环境噪音。揭示分子构象、多样性和动力学的新体外方法可以在生命和化学科学中找到广泛的应用潜力。