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 crystal growth. CLSM is used to study the early crystallization stage of Des-ThrB30 human insulin in aqueous solution, under conditions known to lead to monoclinic crystals. A modified batch crystallization method for CLSM purposes is applied which allows the growth behavior of crystallites to be studied in reflected light. A few hours after the start of the experiment, microcrystallites of characteristic shapes (mainly prismatic and pyramidal) are observed, the number of which strongly depends on the concentration of higher 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 observation times up to 24 h. The anisotropic growt h rates of monoclinic, prismatic crystallites are measured along the long edge of the cover face and perpendicular to the latter. A simultaneous crossover to signifcantly higher growth rates is found when the crystallite size reaches about 2μm. The higher growth rates are connected with the appearence of striations. We argue that this growth rate crossover is caused by an increased 2D nucleation rate at the edges and corners, which finally results in bunching of steps simultaneously spreading over adjacent crystallite faces.