Pseudo-3D Visualization of Cytoskeletal and Whole-Cell Deformation of MLO-Y4 Osteocytes Under Flow

Osteocytes respond to fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell [1] and intracellular deformation [2] may be able to directly activate and modulate relevant biochemical pathways. Most studies on cell deformation have focused only on cell deformation in the plane parallel to the substrate surface. However, height-dependent cell deformation has not been well characterized even though it may contribute greatly to mechanotransduction mechanisms. Traditional techniques to obtain this additional height information of a cell-body include confocal and deconvolution microscopy, which require scanning a z-stack of the cell. However, this inherently limits the timescale under which the deformational information can be visualized. To further investigate this behavior at a high temporal resolution, we propose using a “pseudo-3D” microscopy method to better characterize osteocyte cell behavior. In this study, we present a novel technique that is able to image a single cell simultaneously in two orthogonal planes to obtain real-time images of cell at a millisecond timescale. The objectives of this study were to: (1) visualize actin or microtubule networks with the plasma membrane in two orthogonal planes simultaneously under fluid shear; (2) map out the deformations using digital image correlation; and (3) compare the depth-directional deformation of actin and microtubule networks of osteocytes.