Combined TIRF and 3D Super-Resolution Microscopy for Nanoscopic Characterization of Adhesion Molecules on Microvilli

The homing of hematopoietic stem/progenitor cells (HSPCs) and leukemic cells is a multistep process governed by complex spatiotemporal interactions between adhesion molecules under shear stress. While the molecular and biological mechanisms of this process have been extensively studied, the precise nanoscale spatial organization of adhesion molecules that influences homing efficiency remains relatively poorly understood. In particular, the roles of the cell surface topography and its morphological changes during homing in shaping the spatial organization of adhesion molecules remain elusive. This is partly due to the lack of imaging techniques that simultaneously capture both nanoscopic cell surface morphology and the spatial distribution of the adhesion molecules. Here, we develop a microfluidics-based super-resolution (SR) imaging platform that enables the three-dimensional (3D) mapping of the cell surface morphology and the spatial distribution of the adhesion molecules during HSPC and leukemic cell rolling, which were fixed in situ under shear flow, by integrating total internal reflection fluorescence microscopy (TIRFM) with single-molecule localization microscopy (SMLM). We reconstruct the cell surface morphology, which is critical to the homing, using TIRFM, and precisely overlay the spatial distribution of adhesion molecules, including CD44, PSGL-1, and actin cytoskeleton, determined by 3D-SMLM, on the topographic map. We show distinct nanoscopic localizations of adhesion molecules on the microvilli of HSPCs/leukemic cells and their reorganization under shear stress during cell rolling, at a spatial resolution of approximately 30 nm. The approach offers a powerful means to elucidate the complicated interplay between cell surface morphology and ligand-receptor interactions.