Takuji Adachi

The microscopic understanding of the early stage of crystallization remains a great challenge. The major difficulty in tackling this problem is the stochastic nature of crystallization, from which both experimental and theoretical approaches suffer. A traditional approach to studying crystal nucleation has been measuring the rate of nucleation and modeling the process using classical nucleation theory. Recently, there have been major breakthroughs in both experimental and theoretical approaches to advance understanding of crystal nucleation. The long-awaited moment of “seeing” the instant of crystal nucleation has been realized via microscopies such as in situ and cryo-transmission electron microscopy (TEM). Molecular dynamics (MD) simulations assisted by machine learning have been developed to access the molecular-level details of nucleation processes. Despite these advances, two major questions remain out of reach: what happens just before a crystal nucleus is formed, and when and how a certain polymorph is chosen. A technique that has been underused in probing the early stage of crystallization is optical spectroscopy. The advantages of optical spectroscopy that make it particularly suited for tackling this problem are its sensitivity to structural order and symmetry, its temporal resolution, and the relative ease of experiments and sample preparation. Thus, optical spectroscopy provides information that is complementary to the findings from microscopies and MD simulations. Collectively, we are at an exciting period in this field where we may finally understand the early stage of crystallization and rationally control polymorphism. This perspective highlights recent developments in in situ optical spectroscopy and opportunities awaiting us in the near future.

The formation of crystals has been a long-standing unresolved question while its understanding is crucial for rational design of the crystallization process and controlling polymorphism. Glycine is a well-studied model system as it has been known to form three polymorphs, depending on crystallization conditions. Recently, a new hypothesis has been suggested that glycine forms pre-nucleation aggregates, both in water and saltwater, before nucleating as a metastable β-glycine polymorph. Molecular dynamics simulations proposed the structure of the pre-nucleation aggregates in water as a linear hydrogen-bonded network of glycine molecules. Herein, polarized second harmonic scattering (pol-SHS) was used to test this hypothesis by probing the structural details of glycine aggregates in water and saltwater. Depolarization ratio extracted from a series of concentrations of glycine solutions strongly indicates the presence of aggregates both in water and saltwater and their structure evolves from linear (dipolar) networks to more branched (less dipolar) networks. Retardation ratio of pol-SHS data provided the molecular level details of how water organizes around a glycine molecule/aggregate in water or saltwater. In an aqueous solution, water reorganizes from azimuthal to radial pattern when glycine is dissolved. In saltwater, water organizes in a radial pattern around salt ions and the addition of glycine does not alter the structural organization of water. Glycine aggregates grow while the long-range molecular correlation of the sodium chloride–water networks is maintained. This study demonstrates the potency of pol-SHS in investigating the structure of molecular aggregates in solution in the context of understanding the crystal nucleation mechanism.

Dynamic Light Scattering (DLS) is a powerful technique to probe nano to microscale objects in solution. Among its main limitations is the requirement of steady-state conditions during measurement, restricting its applicability towards time evolving systems. Here, we present a microscopy-based DLS approach using oblique illumination to significantly enhance the signal-to-background ratio compared to conventional confocal geometries. The coherence factor 𝛽 of the autocorrelation function increases to values above 0.9, approaching the ideal limit. Combined with a custom data analysis pipeline enabling post-acquisition processing over arbitrary time windows, this approach yields narrower and better-defined size distributions when averaging over shorter time intervals. Weak scatterers such as BSA proteins can be accurately measured at temporal resolutions down to 20 ms. These results extend the applicability of DLS beyond steady-state conditions and enable time-resolved size determination in dynamic systems.

Stereochemistry controls oligourethane folding through backbone torsions, yielding helices and extended zig-zag structures formed by isotactic and syndiotactic motifs, respectively.

Liquid-liquid phase separation of biomolecules can be induced in vitro by selecting specific concentrations and environmental conditions. Phase diagrams relating the temperature, pressure, etc. to the concentrations of proteins and co-solutes aid in identifying the specific phase separation conditions for target proteins. However, it remains challenging to determining complete phase diagrams for protein liquid-liquid phase separation. Here, we combine optical microscopy and in situ Raman spectroscopy to quantify the temperature-dependent concentrations of the light and dense phases of human γD-crystallin and construct a complete phase diagram. The method can be extended to systems in the presence of co-solutes, which enables the quantitative description of the partitioning of a co-solute in the light and dense phase of proteins.

Raman microspectroscopy offers several advantages over a traditional Raman spectroscopy setup. The major one is its superior signal collection ability owing to the use of high numerical aperture objective lens. The choice of optimal pinholes and high-quality filters also allows one to eliminate unwanted background signals to the least. Such a setup enables one to use a high-power continuous-wave (CW) laser with GHz bandwidth, not MHz, to acquire high-quality Raman spectrum at room temperature. The acquisition of Raman spectrum can therefore be performed with much less acquisition time at a proper home-built Raman microspectroscopy setup. This opens up various applications of Raman spectroscopy toward in situ spectroscopy measurement to identify crystal polymorphs, time-resolved Raman spectroscopy to follow the phase transition of materials, the detection of inorganic nanocrystals in plastic products, etc. Herein, we describe the key practical points to consider in designing and building a setup, and then introduce several applications that have been recently published from our group.

Molecular self-assembly can be a powerful strategy for fabricating dynamic two-dimensional (2D) nanomaterials. However, framing a rational design principle that can satisfy the strict thermodynamic and kinetic criteria enabling a uniform in-plane growth, as well as offer a provision for dynamic structural rearrangement, presents a considerable challenge. We report a hydrophobic core-substituted naphthalenediimide that self-assembles in water-dioxane mixtures to form a pair of 2D poly-morphs, with distinct morphological, optical and surface characteristics. Changing the solvent composition or temperature prompts an in-situ transformation between the two polymorphs in solution. A comprehensive structural investigation using solid-state NMR spectroscopy, x-ray and electron diffraction analyses reveals not only the nature of molecular packing and noncovalent (n-pi* and pi-stacking) interactions stabilizing each 2D polymorph, but also the role of weak solvent-solute interactions in driving polymorphic interconversion. Different arrangements of perfluoroalkyl sidechains at the exposed 2D surface of the two polymorphs result in difference in solvent-solute interactions at the solid-liquid interface, which affects the surface free-energy and relative stability in different solvent compositions. Further, entropy change associated with surface ordering of perfluoroalkyl sidechains and desolvation dictates thermal reorganization between the polymorphs. How changes in dioxane-water microheterogeneity with solution composition and temperature may affect these weak solvent-solute interactions and the phase equilibrium is also discussed.

The electron transfer complex of [FeFe]-hydrogenase and ferredoxin was probed under reducing or oxidizing conditions. Infrared spectroscopy indicates structural changes that modulate redox-dependent protein–protein interaction.