Interferometric scattering (iSCAT) microscopy has demonstrated unparalleled performance among label-free optical methods for detecting and imaging isolated nanoparticles and molecules. However, when imaging complex structures such as biological cells, the superposition of the scattering fields from different locations of the sample leads to a speckle-like background, posing a significant challenge in deciphering fine features. Here, we show that by controlling the spatial coherence of the illumination, one can eliminate the spurious speckle without sacrificing sensitivity. We demonstrate this approach by positioning a rotating diffuser coupled with an adjustable lens and an iris in the illumination path. We report on imaging at a high frame rate of 25 kHz and across a large field of view of 100µm×100µm, while maintaining diffraction-limited resolution. We showcase the advantages of these features by three-dimensional (3D) tracking over 1000 vesicles in a single COS-7 cell and by imaging the dynamics of the endoplasmic reticulum (ER) network. Our approach opens the door to the combination of label-free imaging, sensitive detection, and 3D high-speed tracking using wide-field iSCAT microscopy.

中文翻译：

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iSCAT 显微镜和具有定制空间相干性的粒子跟踪


干涉散射 (iSCAT) 显微镜在用于检测和成像分离纳米颗粒和分子的无标记光学方法中表现出无与伦比的性能。然而，当对生物细胞等复杂结构进行成像时，样品不同位置的散射场的叠加会导致散斑状背景，这对解读精细特征构成了重大挑战。在这里，我们表明，通过控制照明的空间相干性，可以在不牺牲灵敏度的情况下消除杂散散斑。我们通过在照明路径中放置一个旋转扩散器以及一个可调节透镜和光圈来演示这种方法。我们报告了以 25 kHz 的高帧速率和 100μm×100μm 的大视场进行成像，同时保持衍射极限分辨率。我们通过三维 (3D) 跟踪单个 COS-7 细胞中的 1000 多个囊泡并对内质网 (ER) 网络的动态进行成像，展示了这些功能的优势。我们的方法打开了使用宽视场 iSCAT 显微镜将无标记成像、灵敏检测和 3D 高速跟踪相结合的大门。