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.

PPM Detection Limits in PXRD by Integrating Nonlinear Optics and Synchrotron XRD

SHG microscopy allows rapid and selective identification of trace chiral crystals within amorphous media, enabling targeted XRD using a 5-10 micrometer diameter "minibeam". The sensitivity of PXRD is increased substantially by reducing the background scattering contributions of amorphous material otherwise encountered with a larger beam. In addition, performing diffraction only at the locations most likely to produce diffraction greatly reduced the overall beam-time required to perform the PXRD analyses. Integration of the SHG microscope directly into a synchrotron X-ray beamline at Argonne National Laboratory recovered high spatial registry between the regions of interest identified by SHG for positioning within the X-ray beam. Using this approach, diffraction was performed on individual griseofulvin nanocrystals suspended within an amorphous polymer, corresponding to a total of ~20 fg of total crystalline material. Additional measurements for ritonavir in hydroxypropylmethylcellulose (HPMC) were also performed, in which a bulk API concentration of 100 ppm produced diffraction peaks with a signal to noise ratio of >3000. Among other applications, sensitive detection of trace crystallinity can inform the design of amorphous formulations, in which the bioavailability of active pharmaceutical ingredients (APIs) is enhanced by maintaining them in an amorphous state. However, the long-term stability of a final dosage form can be negatively impacted by spontaneous transitioning to the typically more stable crystalline forms of the APIs, such that extensive quantitative characterization of the crystallization behaviors of amorphous formulations is routinely performed.