Light scattering by nanoscale objects is a fundamental physical property defined by their scattering cross-section and thus polarizability. Over the past decade, a number of studies have demonstrated single-molecule sensitivity by imaging the interference between scattering from the object of interest and a reference field. This approach has enabled mass measurement of single biomolecules in solution owing to the linear scaling of image contrast with molecular polarizability. Nevertheless, all implementations so far are based on a common-path interferometer and cannot separate and independently tune the reference and scattered light fields, thereby prohibiting access to the rich toolbox available to holographic imaging. Here we demonstrate comparable sensitivity using a non-common-path geometry based on a dark-field scattering microscope, similar to a Mach–Zehnder interferometer. We separate the scattering and reference light into four parallel, inherently phase-stable detection channels, delivering a five orders of magnitude boost in sensitivity in terms of scattering cross-section over state-of-the-art holographic methods. We demonstrate the detection, resolution and mass measurement of single proteins with mass below 100 kDa. Separate amplitude and phase measurements also yield direct information on sample identity and experimental determination of the polarizability of single biomolecules.

纳米级物体的光散射是由其散射截面和极化率定义的基本物理特性。在过去的十年中，许多研究通过对感兴趣物体和参考场的散射之间的干扰进行成像，证明了单分子灵敏度。由于图像对比度与分子极化率的线性缩放，这种方法能够对溶液中的单个生物分子进行质量测量。然而，迄今为止的所有实现都是基于共路干涉仪，无法分离和独立调谐参考光场和散射光场，从而无法访问可用于全息成像的丰富工具箱。在这里，我们使用基于暗场散射显微镜的非共路径几何结构（类似于马赫-曾德干涉仪）展示了相当的灵敏度。我们将散射光和参考光分成四个平行的、本质上相位稳定的检测通道，与最先进的全息方法相比，散射截面的灵敏度提高了五个数量级。我们演示了质量低于 100 kDa 的单一蛋白质的检测、分辨率和质量测量。单独的幅度和相位测量还可以产生有关样品身份和单个生物分子极化性实验测定的直接信息。