Phase-Shifting Interferometry for the Study of the Step Dynamics during Crystallization of Proteins

摘要

We have developed a novel phase-shifting interferometry technique for high-resolution in-situ investigations of the unsteady dynamics of growth steps during the crystallization of proteins. The phase-shifting algorithm employs five-image sequences captured with a phase shift of π/2; digital processing of the sequence allows reconstruction of the surface morphology with a depth resolution <5 nm and a lateral resolution of 0.5 μm across a field of view as wide as 1 mm. Such sequences can be recorded with a frequency of ～1 s~(-1) and allow monitoring of the appearance and evolution of local morphology features, such as step bunches. Time traces of the variations of the growth rate and local slope (proportional to the density of the growth steps) at up to 10 select locations on a studied crystals facet are recorded with time resolution that can be as low as 0.2 s. Application of this technique to the ferritin crystals shows extensive fluctuations of growth rate and local slope as a result of step bunching.