Single-Molecule Imaging in Commercial Stationary Phase Particles Using Highly Inclined and Laminated Optical Sheet Microscopy

We resolve the three-dimensional, nanoscale locations of single-molecule analytes within commercial stationary phase materials using highly inclined and laminated optical sheet (HILO) microscopy. Single-molecule fluorescence microscopy of chromatography can reveal the molecular heterogeneities that lead to peak broadening, but past work has focused on surfaces designed to mimic stationary phases, which have different physical and chemical properties than the three-dimensional materials used in real columns and membranes. To extend single-molecule measurements to commercial stationary phases, we immobilize individual stationary phase particles and modify our microscope for imaging at further depths with HILO, a method which was originally developed to resolve single molecules in cells of comparable size to column packing materials (∼5–10 μm). We describe and characterize how to change the angle of incidence to achieve HILO so that other researchers can easily incorporate this method onto their existing epi- or total internal reflection fluorescence microscopes. We show improvements up to a 32% in signal-to-background ratio and 118% in the number of single molecules detected within stationary phase particles when using HILO compared to epifluorescence. By controlling the objective position relative to the sample, we produce three-dimensional maps of molecule locations throughout entire stationary phase particles at nanoscale lateral and axial resolutions. The number of localized molecules remains constant axially throughout isolated stationary phase particles and between different particles, indicating that heterogeneity in a separation would not be caused by such affinity differences at microscales but instead kinetic differences at nanoscales on identifiable and distinct adsorption sites.

我们使用高倾角薄层光学片（highly inclined and laminated optical sheet, HILO）显微镜，实现了商业固定相（stationary phase）材料内单分子分析物的三维纳米级定位。色谱单分子荧光显微镜技术可揭示导致色谱峰展宽的分子异质性，但过往研究多聚焦于模拟固定相的表面体系，这类体系与实际色谱柱和膜中使用的三维固定相材料在物理和化学性质上存在差异。为将单分子测量拓展至商业固定相体系，我们固定了单个固定相颗粒，并利用HILO技术改造显微镜以实现更深层成像——该方法最初被开发用于定位与色谱填料尺寸（约5~10 μm）相当的细胞内单分子。我们详细描述并表征了通过调整入射角实现HILO成像的方法，以便其他研究者可轻松将该技术集成至现有落射荧光或全内反射荧光显微镜系统中。相较于落射荧光成像，使用HILO技术时，固定相颗粒内的信噪比最高可提升32%，检测到的单分子数量最多可增加118%。通过调控物镜与样品的相对位置，我们实现了整个固定相颗粒内分子位置的三维测绘，横向和轴向分辨率均达到纳米级。单个固定相颗粒内部以及不同颗粒之间，轴向方向上的定位分子数量均保持恒定，这表明色谱分离中的异质性并非由微观尺度下的亲和性差异导致，而是源于纳米尺度下可识别的不同吸附位点上的动力学差异。