When suitable crystallization conditions are found, characterization of the crystalline sample is an important step to ensure success of a serial crystallography experiment. Several methods are available for identifying (nano)crystals and judging the quality of the crystalline sample.

Light microscopy

The most common technique for characterization of the crystalline sample uses light microscopy. A high spatial resolution (high numerical aperture, good optics and correct alignment of the microscope) is required when looking for microcrystals, which can be achieved with a high-quality optics, a high numerical aperture, and correct alignment of the microscope. However, identifying crystals smaller than 2 µm remains challenging with a light microscope.

Fluorescence microscopy

Intrinsic fluorescence of tryptophan residues in the protein can be used for identifying crystals with fluorescence microscopy. Crystals are excited using 280 nm light, resulting in tryptophan fluorescence at 350 nm. Alternatively, crystals can be soaked with fluorescent dyes (for example in the case your protein of interest does not contain tryptophan). Unfortunately, the method does not distinguish crystals from aggregation.

Second order non-linear imaging of chiral crystals

Second Order Non-linear Imaging of Chiral Crystals (SONICC) is a technique that utilizes the ability of a protein crystal to produce a Second Harmonic Generation (SHG) signal. This property allows for the detection of crystals smaller than 1 µm in almost any environment (including the lipidic cubic phase, for crystallizing membrane proteins). Thin crystalline plates and crystals buried in precipitated protein can also be identified. It should be noted that crystals with high symmetry may not be detected as easily with SHG, due to internal cancellation of the signal.

Reversibility of crystallization

Many techniques for screening microcrystals are based on expensive technology that may not be available in your lab. An economic alternative is testing the reversibility of crystallization, which can help distinguish well-ordered protein crystals from amorphous precipitate. Reversibility of crystallization is based on the idea that nanocrystals dissolve after diluting the drop containing the crystals, whereas denatured, precipitated protein does not. This technique can be performed using just a microscope and pipetting work (or a crystallization robot).

Dynamic light scattering

Dynamic light scattering (DLS) gives information on the size, distribution, and aggregation state of macromolecules in solution, making it a great tool for characterizing crystalline samples. By measuring intensity fluctuations of light scattering by individual molecules, the translational diffusion coefficient and hydrodynamic radius of particles in solution can be calculated. DLS can be used to monitor particle size growth over time to obtain information on nucleation conditions and follow the crystallization process.