Justin A. Newman

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction for combined single-platform analysis, examining the viability of NLO microscopy as an alternative to the conventional X-ray raster scan for the purposes of sample centering. Second-harmonic generation microscopy and two-photon excited ultraviolet fluorescence microscopy were evaluated for crystal detection, and assessed by X-ray raster scanning.

High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories

A simple beam-scanning optical design based on Lissajous trajectory imaging is described for achieving up to kHz frame-rate optical imaging on multiple simultaneous data acquisition channels. In brief, two fast-scan resonant mirrors direct the optical beam on a circuitous trajectory through the field of view, with the trajectory repeat-time given by the least common multiplier of the mirror periods. Dicing the raw time-domain data into sub-trajectories combined with model-based image reconstruction (MBIR) 3D in-painting algorithms allows for effective frame-rates much higher than the repeat time of the Lissajous trajectory. Since sub-trajectory and full-trajectory imaging are simply different methods of analyzing the same data, both high-frame rate images with relatively low resolution and low frame rate images with high resolution are simultaneously acquired. The optical hardware required to perform Lissajous imaging represents only a minor modification to established beam-scanning hardware, combined with additional control and data acquisition electronics. Preliminary studies based on laser transmittance imaging and polarization-dependent second harmonic generation microscopy support the viability of the approach both for detection of subtle changes in large signals and for trace-light detection of transient fluctuations.

Parts per Million Powder X-ray Diffraction

Here we demonstrate the use of second harmonic generation (SHG) microscopy-guided synchrotron powder X-ray diffraction (PXRD) for the detection of trace crystalline active pharmaceutical ingredients in a common polymer blend. The combined instrument is capable of detecting 100 ppm crystalline ritonavir in an amorphous hydroxypropyl methylcellulose matrix with a high signal-to-noise ratio (>5000). The high spatial resolution afforded by SHG microscopy allows for the use of a minibeam collimator to reduce the total volume of material probed by synchrotron PXRD. The reduction in probed volume results in reduced background from amorphous material. The ability to detect low crystalline loading has the potential to improve measurements in the formulation pipeline for pharmaceutical solid dispersions, for which even trace quantities of crystalline active ingredients can negatively impact the stability and bioavailability of the final drug product.

Guiding synchrotron X‐ray diffraction by multimodal video‐rate protein crystal imaging

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s(-1)). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-fold improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.

Dynamic X-ray diffraction sampling for protein crystal positioning

A sparse supervised learning approach for dynamic sampling (SLADS) is described for dose reduction in diffraction-based protein crystal positioning. Crystal centering is typically a prerequisite for macromolecular diffraction at synchrotron facilities, with X-ray diffraction mapping growing in popularity as a mechanism for localization. In X-ray raster scanning, diffraction is used to identify the crystal positions based on the detection of Bragg-like peaks in the scattering patterns; however, this additional X-ray exposure may result in detectable damage to the crystal prior to data collection. Dynamic sampling, in which preceding measurements inform the next most information-rich location to probe for image reconstruction, significantly reduced the X-ray dose experienced by protein crystals during positioning by diffraction raster scanning. The SLADS algorithm implemented herein is designed for single-pixel measurements and can select a new location to measure. In each step of SLADS, the algorithm selects the pixel, which, when measured, maximizes the expected reduction in distortion given previous measurements. Ground-truth diffraction data were obtained for a 5 µm-diameter beam and SLADS reconstructed the image sampling 31% of the total volume and only 9% of the interior of the crystal greatly reducing the X-ray dosage on the crystal. Using in situ two-photon-excited fluorescence microscopy measurements as a surrogate for diffraction imaging with a 1 µm-diameter beam, the SLADS algorithm enabled image reconstruction from a 7% sampling of the total volume and 12% sampling of the interior of the crystal. When implemented into the beamline at Argonne National Laboratory, without ground-truth images, an acceptable reconstruction was obtained with 3% of the image sampled and approximately 5% of the crystal. The incorporation of SLADS into X-ray diffraction acquisitions has the potential to significantly minimize the impact of X-ray exposure on the crystal by limiting the dose and area exposed for image reconstruction and crystal positioning using data collection hardware present in most macromolecular crystallography end-stations.

Imaging local electric fields produced upon synchrotron X-ray exposure.

Significance Exposure to high-energy X-rays, experienced during X-ray diffraction experiments for determining atomic structures, can potentially create large local electric fields from photoelectron ejection. Under cryogenic conditions, these fields may persist indefinitely. Mapping of electric fields is important for a complete understanding of damage mechanisms involved in X-ray measurements, for potential effects on the quality and interpretation of X-ray data, and for possible direct impact on diffraction resolution through the piezoelectric effect. Mapping of electric fields may also be useful in retaining a record of locations of X-ray exposure. In this paper, electric-field induced second harmonic generation imaging is explored in simulation and experiment. It provides insight into the position and distribution of local electric fields within an X-ray exposed sample. Electron–hole separation following hard X-ray absorption during diffraction analysis of soft materials under cryogenic conditions produces substantial local electric fields visualizable by second harmonic generation (SHG) microscopy. Monte Carlo simulations of X-ray photoelectron trajectories suggest the formation of substantial local electric fields in the regions adjacent to those exposed to X-rays, indicating a possible electric-field–induced SHG (EFISH) mechanism for generating the observed signal. In studies of amorphous vitreous solvents, analysis of the SHG spatial profiles following X-ray microbeam exposure was consistent with an EFISH mechanism. Within protein crystals, exposure to 12-keV (1.033-Å) X-rays resulted in increased SHG in the region extending ∼3 μm beyond the borders of the X-ray beam. Moderate X-ray exposures typical of those used for crystal centering by raster scanning through an X-ray beam were sufficient to produce static electric fields easily detectable by SHG. The X-ray–induced SHG activity was observed with no measurable loss for longer than 2 wk while maintained under cryogenic conditions, but disappeared if annealed to room temperature for a few seconds. These results provide direct experimental observables capable of validating simulations of X-ray–induced damage within soft materials. In addition, X-ray–induced local fields may potentially impact diffraction resolution through localized piezoelectric distortions of the lattice.

Unified Theory for Polarization Analysis in Second Harmonic and Sum Frequency Microscopy

A unified theoretical framework for the recovery of second-order nonlinear susceptibility tensors and sample orientations from polarization-dependent second harmonic generation and sum frequency generation microscopy was developed. Jones formalism was extended to nonlinear optics and was used to bridge the experimental observables and the local-frame tensor elements. Four commonly used experimental architectures were explicitly explored, including polarization rotation with no postsample optics, polarization-in polarization-out measurement, and polarization modulation with and without postsample optics. Polarization-dependent second harmonic generation measurement was performed on Z-cut quartz and the local-frame tensor elements were calculated. The recovered tensor elements agree with the expected values dictated by symmetry.

Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals.

The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy.

Sparse sampling image reconstruction in Lissajous trajectory beam-scanning multiphoton microscopy

Propagation of action potentials arises on millisecond timescales, suggesting the need for advancement of methods capable of commensurate volume rendering for in vivo brain mapping. In practice, beam-scanning multiphoton microscopy is widely used to probe brain function, striking a balance between simplicity and penetration depth. However, conventional beam-scanning platforms generally do not provide access to full volume renderings at the speeds necessary to map propagation of action potentials. By combining a sparse sampling strategy based on Lissajous trajectory microscopy in combination with temporal multiplexing for simultaneous imaging of multiple focal planes, whole volumes of cells are potentially accessible each millisecond.

Multi-channel beam-scanning imaging at kHz frame rates by Lissajous trajectory microscopy.

A beam-scanning microscope based on Lissajous trajectory imaging is described for achieving streaming 2D imaging with continuous frame rates up to 1.4 kHz. The microscope utilizes two fast-scan resonant mirrors to direct the optical beam on a circuitous trajectory through the field of view. By separating the full Lissajous trajectory time-domain data into sub-trajectories (partial, undersampled trajectories) effective frame-rates much higher than the repeat time of the Lissajous trajectory are achieved with many unsampled pixels present. A model-based image reconstruction (MBIR) 3D in-painting algorithm is then used to interpolate the missing data for the unsampled pixels to recover full images. The MBIR algorithm uses a maximum a posteriori estimation with a generalized Gaussian Markov random field prior model for image interpolation. Because images are acquired using photomultiplier tubes or photodiodes, parallelization for multi-channel imaging is straightforward. Preliminary results show that when combined with the MBIR in-painting algorithm, this technique has the ability to generate kHz frame rate images across 6 total dimensions of space, time, and polarization for SHG, TPEF, and confocal reflective birefringence data on a multimodal imaging platform for biomedical imaging. The use of a multichannel data acquisition card allows for multimodal imaging with perfect image overlay. Image blur due to sample motion was also reduced by using higher frame rates.