In Situ Observation of Elementary Growth Processes of Protein Crystals by Advanced Optical Microscopy

Gen      Sazaki; Alexander      E.S. Van Driessche; Guoliang      Dai; Masashi      Okada; Takuro      Matsui; Fermin      Otalora; Katsuo      Tsukamoto; Kazuo      Nakajima

Abstract

To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

Keywords: Adsorption, advanced optical microscopy, elementary steps, impurity effect, in situ observation, single molecule visualization, surface diffusion, two-dimensional nucleation, Atomic force microscopy , LCM-DIM