Second-order nonlinear optical imaging of chiral crystals

Abstract

Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald's rule of stages for crystal growth. SONICC's selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation.

Figure 1

(a) Time-domain distortion of the induced polarization from anharmonicity in the molecular polarizability. (b) The corresponding frequency contributions combine to recover the distortion.

Figure 2

Visual hyperellipsoid representation of the molecular tensor (selected arbitrarily) and illustration of the symmetry operations within a crystal lattice. The symmetry operations of a crystal in the P2₁2₁2₁ space group yield the net hyperellipsoid representation shown at the bottom.

Figure 3

General instrument schematic for SONICC (second-order nonlinear optical imaging of chiral crystals) measurements. Abbreviations: DM, dichroic mirror; PMT, photomultiplier tube.

Figure 4

Screen shots of NLOPredict, a program designed to help interpret polarization-dependent nonlinear optical (NLO) measurements of biopolymer and small-molecule assemblies (shown for Protein Data Bank entry 2RH1).

Figure 5

Comparison of SONICC (second-order nonlinear optical imaging of chiral crystals) and conventional optical methods for protein-crystal detection, from six representative outcomes of crystallization trials (a–f). Bright-field microscopy and birefringence (cross-polarized) images were obtained through the use of white-light illumination with UV and Cy3 fluorescence excited by ~280-nm and ~543-nm light, respectively. All SONICC images were acquired with 800-nm incident light with detection at 400 nm. The images in panels c and f appear identical according to all four conventional methods. SONICC analysis reveals that the image in panel c contains a shower of microcrystals (~2 µm or less in diameter), whereas that in panel f is completely clear. The presence of small crystals can be determined in trials that are highly turbid (b,e). The dotted lines indicate the origin of the line traces in Figure 6. Abbreviation: N/A, not applicable.

Figure 6

Line traces, corresponding to the dashed lines in Figure 5b, comparing UV fluorescence and SONICC (second-order nonlinear optical imaging of chiral crystals) for detection of unlabelled protein crystals prepared in meso. Whereas the intrinsic UV enables detection of one crystal with a moderate signal-to-background ratio, SONICC clearly indicates the presence of at least five crystals spanning the line trace. Even in the clear positive case shown in Figure 5a, the signal-to-background ratios for UV fluorescence, Cy3 fluorescence, and SONICC are 1.5:1, 5:1, and 12,000:1, respectively.

Figure 7

Comparison between manual scoring and automated scoring for different sets of images. The graph is divided into four regions on the basis of the minimum score that was considered a positive in each case. Color coding and shapes indicate manual categorization based on inspection of the SONICC (second-order nonlinear optical imaging of chiral crystals) images.

Figure 8

(a) Real-time crystallization of griseofulvin monitored by SONICC (second-order nonlinear optical imaging of chiral crystals). Individual crystals (circled in red) can be analyzed using standard threshold-based particle analysis. (b) Isothermal crystallization kinetics of griseofulvin probed by SONICC. The overall crystallization rate, nucleation rate, and growth rate of individual crystals can be simultaneously analyzed.

Figure 9

Hypothetical free-energy curves highlighting the possibility of polymorph curve crossing during crystallization. Solid lines represent the net free energy as a function of the cluster size, generated from the sum of the surface area and volume terms (dashed lines). The critical cluster size for nucleation, N^‡, is indicated by asterisks.

Figure 10

(a) SONICC (second-order nonlinear optical imaging of chiral crystals) images of transient second harmonic generation–active crystallites (red circles) during solvent evaporation of an aqueous serine solution. (b) Similar measurements performed with a homochiral solution. Time zero was defined by the presence of detectable SONICC signals, which corresponded to the onset of crystal formation. Panel b has been rescaled by a factor of two to promote comparison. Images adapted from Reference .