A Novel Technique of Quantifying Flexural Stiffness of Rod-Like Structures

In cellular and molecular biomechanics, extensional stiffness of rod-like structures such as leukocyte microvilli can be easily measured with many techniques, but not many techniques are available for measuring their flexural stiffness. In this paper, we report a novel technique of measuring the flexural stiffness of rod-like structures. This technique is based on image deconvolution and, as an example, it was used for determining the flexural stiffness of neutrophil microvilli. The probes we used were 40-nm-diameter fluorescent beads, which were bound to the tips of neutrophil microvilli by anti-L-selectin antibody. The fluorescent images of the bead, which was positioned at the center of the cell bottom, were acquired with high magnification and long exposure time (3 s). Using a Gaussian function as the point spread function of our imaging system, we established a convolution equation based on Boltzmann's law, which yields an analytical expression that relates the bead image profile to the flexural stiffness of the microvillus. The flexural stiffness was then obtained by the least squares regression. On average, the flexural stiffness was determined to be 7 pN/mum for single neutrophil microvilli. With the resolution of our imaging system, this technique can be used for measuring any flexural stiffness smaller than 34 pN/mum and it has great potential in single molecule biomechanics.