Quantifying bio-filament morphology below the diffraction limit of an optical microscope using out-of-focus images.

A method to measure the dimensions of objects below the optical diffraction limit using diffraction analysis of out-of-focus bright-field images is presented. The method relies on the comparison of the diffraction patterns of an object of unknown size to those of calibration objects of known size. Correlative scanning electron microscope measurements are used to demonstrate the applicability of this method to measure 100 nm microbeads as well as objects with a geometry different from the calibration objects. This technique is important in the context of tethered particle experiments, in which bio-filaments are bound between a substrate and a microbead. This procedure is applied to obtain the diameters of axonal extensions or neurites that are mechanically created in samples of rat hippocampal neurons. The dependence of neurite geometry on mechanical pull speed is investigated, and the diameter is found to be rate independent.