Observation of the early stage of insulin crystallization by confocal laser scanning microscopy

It is demonstrated that high resolution confocal laser scanning microscopy (CLSM) is a powerful tool for in situ observation and analysis of protein c rystal growth. CLSM is used to study the early crystallization stage of Des -ThrB30 human insulin in aqueous solution, under conditions known to lead t o monoclinic crystals. A modified batch crystallization method for CLSM pur poses is applied which allows the growth behavior of crystallites to be stu died in reflected light. A few hours after the start of the experiment, mic rocrystallites of characteristic shapes (mainly prismatic and pyramidal) ar e observed, the number of which strongly depends on the concentration of hi gher insulin aggregates in the initial solution. From direct observation as well as from model calculations we conclude that for solute concentrations up to about 3.5-times the saturation value, growth starts from few active insulin precipitate particles while 3D nucleation is neglegible for observa tion times up to 24h. The anisotropic growth rates of monoclinic, prismatic crystallites are measured along the long edge of the cover face and perpen dicular to the latter. A simultaneous crossover to significantly higher gro wth rates is found when the crystallite size reaches about 2 mum. The highe r growth rates are connected with the appearence of striations. We argue th at this growth rate crossover is caused by an increased 2D nucleation rate at the edges and corners, which finally results in bunching of steps simult aneously spreading over adjacent crystallite faces. (C) 2001 Elsevier Scien ce B.V. All rights reserved.