Second-order Nonlinear Optical Imaging Of Chiral Crystals Research Articles

Towards User Operations of SONICC at GM/CA@APS

Second order nonlinear optical imaging of chiral crystals (SONICC), based on femtosecond laser scanning microscopy, has been implemented at GM/CA@APS undulator beamline 23ID-B for rapid protein crystal localization and centering. The technique is based on infrared laser light impinging on non-centrosymmetric crystals of proteins, which selectively may yield a frequency-doubled, visible signal generated by the anharmonic response of the electron cloud of the protein in response to the laser field. One aim of this method is to locate small crystals grown in opaque crystallization media for centering in X-ray beams of only a few microns or less in cross-section [1]. The optical system implemented at the beamline includes `trans' and `epi' detection of Second Harmonic Generation (SHG) signals [2]. In addition, scanning visible laser light across the sample and detecting two-photon excited UV fluorescence (TPE-UVF) provides complementary contrast based on the native fluorescence of proteins. An update on progress towards offering a user-friendly system to users will be provided. Different factors that influence imaging signals and the practice of successfully locating and accurately positioning a crystal via SONICC will be discussed.

Crystallization and Dissolution Behavior of Naproxen/Polyethylene Glycol Solid Dispersions

The crystallization kinetics of naproxen (NAP) in NAP/polyethylene glycol (NAP/PEG) solid dispersions prepared at different crystallization temperatures was studied by in situ small-angle X-ray scattering/wide-angle X-ray scattering (SAXS/WAXS). It was found that the crystallization rate of NAP was faster at 25 °C in comparison to 40 °C. This resulted in different sizes of NAP domains, and consequently impacted the dissolution behavior. The sizes of NAP domains prepared at 40 °C were larger than those at 25 °C, as determined with surface area analysis, utilizing second-order nonlinear optical imaging of chiral crystals (SONICC). Consistent with this observation, the corresponding dissolution rate of the NAP/PEG dispersion prepared at 40 °C was indeed slower than that prepared at 25 °C. The microstructure of the NAP/PEG solid dispersions and the dissolution behavior also showed a dependence on the chemical composition of the solid dispersions.

Selective imaging of active pharmaceutical ingredients in powdered blends with common excipients utilizing two-photon excited ultraviolet-fluorescence and ultraviolet-second order nonlinear optical imaging of chiral crystals.

Second order nonlinear optical imaging of chiral crystals (SONICC) and two-photon excited fluorescence measurements [both autofluorescence and two-photon excited UV-fluorescence (TPE-UVF)] were assessed for the selective detection of APIs relative to common pharmaceutical excipients. Active pharmaceutical ingredients (APIs) compose only a small percentage of most tabulated formulations, yet the API distribution within the tablet can affect drug release and tablet stability. Complementary measurements using either UV-SONICC (266 nm detection) or TPE-UVF were shown to generate signals >50-fold more intense for a model API (griseofulvin) than those produced by common pharmaceutical excipients. The combined product of the measurements produced signals >10(4)-fold greater than the excipients studied. UV-SONICC or TPE-UVF produced greater selectivity than analogous measurements with visible-light detection, attributed to the presence of aromatic moieties within the API exhibiting strong one and two photon absorption at ~266 nm. Complementary SONICC and fluorescence measurements allowed for the sensitive detection of the three-dimensional distribution of tadalafil within a Cialis tablet to a depth of >140 μm.

Imaging of Protein Crystals with Two-Photon Microscopy

Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials.

Two-photon excited UV fluorescence for protein crystal detection

Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging ofchiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

Crystal screening by second-order nonlinear optical imaging of chiral crystals (SONICC)

Crystal screening by second-order nonlinear optical imaging of chiral crystals (SONICC)

Second-order nonlinear optical imaging of chiral crystals.

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.

Single Particle Nonlinear Optical Imaging of Trace Crystallinity in an Organic Powder

Microscopic characterization of crystallinity in powders can reveal information lost in ensemble-averaged measurements. Nonlinear optical imaging based on second harmonic generation (SHG) provides rapid and highly selective detection of individual chiral microcrystals, enabling insights into the fundamental mechanism of action for the observed crystallinity loss of an organic powder induced by mechanical grinding. Using griseofulvin as the model compound, the results from second order nonlinear optical imaging of chiral crystals (SONICC) compared favorably with those of powder X-ray diffraction (PXRD) over the linear dynamic range of the PXRD measurements. However, the SHG measurements demonstrated three decade improvements in linear dynamic range. The detection limit of SHG was estimated to be 4 ppm crystallinity in the powder. The rate of crystallinity loss induced by milling followed a first order process with a half-life of 15 ± 1 min. Recrystallization of cryomilled powder is ~40 times faster than that prepared by melt-quenched powder, suggesting that the disordered state obtained by exhaustive cryomilling appears to contain ordered domains that are larger than the critical nucleation size, but below the detection limit of SONICC. The presence of such domains provides a barrier-less nucleation source resulting in rapid crystallization, the kinetics of which depends only on crystal growth.

Direct Observation of Transient Ostwald Crystallization Ordering from Racemic Serine Solutions

The Ostwald rule of stages describes the conjectured transitioning through metastable polymorphic crystal structures during crystallization. Direct observation of the Ostwald rule of stages using was performed using solutions of simple amino acids by second-order nonlinear optical imaging of chiral crystals (SONICC). SONICC, which is based on second-harmonic generation (SHG) imaging, enabled detection of homochiral microcrystals that survived only a few seconds before being converted to the more stable SHG-inactive polymorphic forms.

Selective Detection and Quantitation of Organic Molecule Crystallization by Second Harmonic Generation Microscopy

Second order nonlinear optical imaging of chiral crystals (SONICC) was applied to selectively detect crystal formation at early stages and characterize the kinetics of nucleation and growth. SONICC relies on second harmonic generation (SHG), a nonlinear optical effect that only arises from noncentosymmetric ordered domain structures, which include crystals of chiral molecules. The model systems studied include pharmaceutically relevant compounds: griseofulvin and chlorpropamide. SONICC demonstrates low detection limits producing an 8 order of magnitude improvement relative to macroscopic average techniques and 5 order of magnitude improvement relative to optical microscopy. SONICC was also applied to examine the kinetics of crystallization in amorphous griseofulvin. The results show that SONICC enables simultaneous monitoring of individual crystal growth, nucleation rate, and macroscopic crystallization kinetics.

Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.

Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 mum) with a minimal background due to the surrounding lipid mesophase ( approximately 1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.

Associate Professor of Chemistry

Second-order nonlinear optical imaging of chiral crystals (SONICC) enables sensitive and selective detection of sub-diffraction limited protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. Under low magnificaiton (large field of view) applications, SONICC can detect individual crystals as small as 100 nm in diameter, which is conservatively 6 orders of magnitude lower that achievalbe using current methods for crystal detection in commercial screening platforms. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets using a crystallization micro-array, the SHG intensities rivaled those of fluorescence but with superb selectivity for crystalline regions. Furthermore, SONICC is directly compatibility with virtually all common protein crystallization platforms without modification.

Selective Detection of Protein Crystals by Second Harmonic Microscopy

The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.