Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used timeresolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.nucleation | protein | lysozyme | transmission electron microscopy | in situ observation C rystallization can be divided into two processes: nucleation and crystal growth. The crystal growth process has been well examined for a long time, yet the nucleation process is not understood; for example, the nucleation rate of crystals provides a textbook example of order-of-magnitude discrepancies between theoretical predictions and experimental results. Recent proposals have attributed these discrepancies to a nonclassical nucleation pathway, along which a structured crystalline embryo forms within a highly concentrated disordered precursor (1). This mechanism was first proposed for protein crystals (2, 3). Direct observations have demonstrated its applicability to organic (4), inorganic (5, 6), and colloidal (7) crystals. In proteins, clusters of protein molecules have been suggested as precursors; these clusters have mesoscopic sizes from several tens to several hundreds of nanometers and are considered to behave like liquids. It has also been suggested that the precursor is thermodynamically stable with respect to the mother liquid phase but is metastable or unstable with respect to the crystalline phase. The latter nature of the precursor differs from the stable macroscopically dense liquid formed as a result of the liquid-liquid phase separation (8). Such protein-rich mesoscopic clusters have been observed for many proteins, primarily using optical techniques, and have been tentatively identified as precursors for crystal nucleation (9-12). Several important questions concerning this mechanism remain unanswered. First, are the observed mesoscopic clusters actually liquid-like or solid-amorphous? Second, do they play an active role in crystal nucleation? In addition, finally, do the clusters serve as classical heteroge...

Samples of the carbonaceous asteroid Ryugu were brought to Earth by the Hayabusa2 spacecraft. We analyzed seventeen Ryugu samples measuring 1-8 mm. CO 2 -bearing water inclusions are present within a pyrrhotite crystal, indicating that Ryugu’s parent asteroid formed in the outer Solar System. The samples contain low abundances of materials that formed at high temperatures, such as chondrules and Ca, Al-rich inclusions. The samples are rich in phyllosilicates and carbonates, which formed by aqueous alteration reactions at low temperature, high pH, and water/rock ratios < 1 (by mass). Less altered fragments contain olivine, pyroxene, amorphous silicates, calcite, and phosphide. Numerical simulations, based on the mineralogical and physical properties of the samples, indicate Ryugu’s parent body formed ~ 2 million years after the beginning of Solar System formation.

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We observed two-dimensional (2D) nucleation behavior on {110} and {101} faces of tetragonal crystals of model protein lysozyme by laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM). We measured, for the first time directly and noninvasively, the 2D nucleation rates using 99.99% pure lysozyme, 98.5% pure lysozyme (Seikagaku Co.), and 99.99% pure lysozyme with intentionally added impure proteins (fluorescent-labeled lysozyme, covalently bonded dimer of lysozyme, and 18 kDa polypeptide). We found that 2D nucleation was the dominant growth mechanism under conditions adopted in this study, and the 2D nucleation occurred randomly on the entire crystal surface irrespective of supersaturation within the range of σ ) ln(C/C e ) ) 0-1.4, where C is a bulk lysozyme concentration and C e the solubility (crystal size: 0.2-0.3 mm). Repeated 2D nucleation, which continued for 3-4 layers, was also observed mainly when the impure proteins were present. In addition, multilayered 2D islands were formed after the adsorption of relatively large foreign particles on the crystal surface. From the comparison between the 2D nucleation rates determined on the {110} faces with and without the impure proteins, we concluded that homogeneous 2D nucleation occurred under a higher supersaturation range (σ > 0.8), irrespective of the presence of the impurities. In contrast, under a lower supersaturation range (σ < 0.8), we found that significant heterogeneous 2D nucleation dominated the growth mainly when the impure proteins were present. The {101} faces exhibited more significant heterogeneous 2D nucleation induced by smaller amounts of impurities than in the case of the {110} faces. We also determined the ledge free energies of the homogeneous and heterogeneous nucleation. Within the experimental conditions used in this study, we could not find significant dependence of the ledge free energies of the heterogeneous nucleation on the kinds of impure proteins.

A kinetic model is developed to describe the growth of crystals under the influence of foreign particles in terms of heterogeneous two-dimensional nucleation. In the context of this model, the free energy barrier of two-dimensional nucleation in the presence of foreign particles and the kinetics for the nucleation and growth are examined theoretically. It follows that the contact angle, size and density of adsorbed foreign particles play a crucial role in controling the 2D nucleation barrier and growth kinetics. Based on our model, many crucial experimental findings, such as dust-induced surface roughening and the various kinetics of dislocation-free growth, are properly interpreted. The promotion effect of foreign particles on crystal growth is also analyzed from the view of designing additives. To our knowledge, this is the first systematic consideration of effects of foreign particles on the 2D nucleation process and growth kinetics, and the model generally covers both heterogeneous and conventional homogeneous 2D nucleation growth.