Visualization of Near Surface Flow of Endothelial Cells based on Confocal Micro-PIV and Super-resolution Microscopy

A glycocalyx layer located on the surface of vascular endothelial cells (ECs), whose thickness was to be 20 nm 4.5 μm measured by transmission electron microscopy, is considered to play an important role in the sensing of fluid force. It is necessary to clarify an influence of the glycocalyx layer on the near surface flow field. However, it is difficult to measure the flow field near the layer because of lack of spatial resolution and accuracy. In this study, we developed a novel visualization technique which has high spatial resolution and high accuracy to investigate the influence of the glycocalyx layer on the near-surface flow field. A measurement technique for three dimensional velocity distributions with spatial resolution of 250 nm in the depth direction was developed by utilizing the confocal micro-PIV technique with optimization of the measurement parameter such as the objective lens, optical filters and by utilizing 100nm silica particles coated with polyethylene glycol (PEG). Super-resolution microscopy was used for the measurement of position and thickness of a glycocalyx layer with lateral resolution of 80 nm. The technique was applied to near surface flow field around EC cultured in a microchannel with a width of 400 μm and depth of 100 μm. The height of the EC and the thickness of the layer around the top of the EC were measured to be 4.67 and 1.33 μm. The velocities near the surface of the glycocalyx layer and the dimension of the layer were successfully measured. The results indicated that the measured velocity distributions show the influence of the layer on the near-surface flow field.