Department of Physics, Chalmers University of Technology, Göteborg, Sweden.
Department of Physics, University of Gothenburg, Göteborg, Sweden.
Nat Methods. 2022 Jun;19(6):751-758. doi: 10.1038/s41592-022-01491-6. Epub 2022 May 30.
Label-free characterization of single biomolecules aims to complement fluorescence microscopy in situations where labeling compromises data interpretation, is technically challenging or even impossible. However, existing methods require the investigated species to bind to a surface to be visible, thereby leaving a large fraction of analytes undetected. Here, we present nanofluidic scattering microscopy (NSM), which overcomes these limitations by enabling label-free, real-time imaging of single biomolecules diffusing inside a nanofluidic channel. NSM facilitates accurate determination of molecular weight from the measured optical contrast and of the hydrodynamic radius from the measured diffusivity, from which information about the conformational state can be inferred. Furthermore, we demonstrate its applicability to the analysis of a complex biofluid, using conditioned cell culture medium containing extracellular vesicles as an example. We foresee the application of NSM to monitor conformational changes, aggregation and interactions of single biomolecules, and to analyze single-cell secretomes.
无标记单分子特征分析旨在补充荧光显微镜，在某些情况下，标记会影响数据分析解读，或者在技术上具有挑战性，甚至是不可能的。然而，现有的方法需要被研究的物种与表面结合才能被观察到，从而导致很大一部分分析物无法被检测到。在这里，我们提出了纳米流控散射显微镜（NSM），它通过允许在纳米流控通道内扩散的单个生物分子进行无标记、实时成像，克服了这些限制。NSM 可以从测量的光学对比度中准确确定分子量，并从测量的扩散系数中确定流体力学半径，从而可以推断出关于构象状态的信息。此外，我们还展示了它在分析复杂生物流体中的应用，以条件细胞培养基（其中含有细胞外囊泡）为例。我们预计 NSM 将可用于监测单分子的构象变化、聚集和相互作用，并分析单细胞分泌组。
