Imaging the Nonlinear Susceptibility Tensor of Collagen by Nonlinear Optical Stokes Ellipsometry.

Nonlinear optical Stokes ellipsometric (NOSE) microscopy was demonstrated for the analysis of collagen-rich biological tissues. NOSE is based on polarization-dependent second harmonic generation imaging. NOSE was used to access the molecular-level distribution of collagen fibril orientation relative to the local fiber axis at every position within the field of view. Fibril tilt-angle distribution was investigated by combining the NOSE measurements with ab initio calculations of the predicted molecular nonlinear optical response of a single collagen triple helix. The results were compared with results obtained previously by scanning electron microscopy, nuclear magnetic resonance imaging, and electron tomography. These results were enabled by first measuring the laboratory-frame Jones nonlinear susceptibility tensor, then extending to the local-frame tensor through pixel-by-pixel corrections based on local orientation.

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.

Video-rate two-photon excited fluorescence lifetime imaging system with interleaved digitization.

A fast (up to video rate) two-photon excited fluorescence lifetime imaging system based on interleaved digitization is demonstrated. The system is compatible with existing beam-scanning microscopes with minor electronics and software modification. Proof-of-concept demonstrations were performed using laser dyes and biological tissue.

Kinetic trapping of metastable amino acid polymorphs.

Second harmonic generation (SHG) microscopy measurements indicate that inkjet-printed racemic solutions of amino acids can produce nanocrystals trapped in metastable polymorph forms upon rapid solvent evaporation. Polymorphism impacts the composition, distribution, and physico-kinetic properties of organic solids, with energetic arguments favoring the most stable polymorph. In this study, unfavored noncentrosymmetric crystal forms were observed by SHG microscopy. Polarization-dependent SHG measurement and synchrotron X-ray microdiffraction analysis of individual printed drops are consistent with formation of homochiral crystal production. Fundamentally, these results provide evidence supporting the ubiquity of Ostwald's Rule of Stages, describing the hypothesized transitioning of crystals between metastable polymorphic forms in the early stages of crystal formation. Practically, the presence of homochiral metastable forms has implications on chiral resolution and on solid form preparations relying on rapid solvent evaporation.

Digital deconvolution filter derived from linear discriminant analysis and application for multiphoton fluorescence microscopy.

A digital filter derived from linear discriminant analysis (LDA) is developed for recovering impulse responses in photon counting from a high speed photodetector (rise time of ~1 ns) and applied to remove ringing distortions from impedance mismatch in multiphoton fluorescence microscopy. Training of the digital filter was achieved by defining temporally coincident and noncoincident transients and identifying the projection within filter-space that best separated the two classes. Once trained, data analysis by digital filtering can be performed quickly. Assessment of the reliability of the approach was performed through comparisons of simulated voltage transients, in which the ground truth results were known a priori. The LDA filter was also found to recover deconvolved impulses for single photon counting from highly distorted ringing waveforms from an impedance mismatched photomultiplier tube. The LDA filter was successful in removing these ringing distortions from two-photon excited fluorescence micrographs and through data simulations was found to extend the dynamic range of photon counting by approximately 3 orders of magnitude through minimization of detector paralysis.

Polarization-modulated second harmonic generation ellipsometric microscopy at video rate.

Fast 8 MHz polarization modulation coupled with analytical modeling, fast beam-scanning, and synchronous digitization (SD) have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and polarized laser transmittance imaging with image acquisition rates up to video rate. In contrast to polarimetry, in which the polarization state of the exiting beam is recorded, NOSE enables recovery of the complex-valued Jones tensor of the sample that describes all polarization-dependent observables of the measurement. Every video-rate scan produces a set of 30 images (10 for each detector with three detectors operating in parallel), each of which corresponds to a different polarization-dependent result. Linear fitting of this image set contracts it down to a set of five parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the incident beam. These parameters can in turn be used to recover the Jones tensor elements of the sample. Following validation of the approach using z-cut quartz, NOSE microscopy was performed for microcrystals of both naproxen and glucose isomerase. When weighted by the measurement time, NOSE microscopy was found to provide a substantial (>7 decades) improvement in the signal-to-noise ratio relative to our previous measurements based on the rotation of optical elements and a 3-fold improvement relative to previous single-point NOSE approaches.

Linear fitting of multi-threshold counting data with a pixel-array detector for spectral X-ray imaging.

Experiments and modeling are described to perform spectral fitting of multi-threshold counting measurements on a pixel-array detector. An analytical model was developed for describing the probability density function of detected voltage in X-ray photon-counting arrays, utilizing fractional photon counting to account for edge/corner effects from voltage plumes that spread across multiple pixels. Each pixel was mathematically calibrated by fitting the detected voltage distributions to the model at both 13.5 keV and 15.0 keV X-ray energies. The model and established pixel responses were then exploited to statistically recover images of X-ray intensity as a function of X-ray energy in a simulated multi-wavelength and multi-counting threshold experiment.

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.

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains.

Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.

Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners.

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.

Assessing cytochrome P450 and UDP-glucuronosyltransferase contributions to warfarin metabolism in humans.

As a step toward exploring a targeted metabolomics approach to personalized warfarin (Coumadin) therapy, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of quantifying specific enantiomeric (R and S) contributions of warfarin (WAR) and the corresponding hydroxywarfarins (OH-WAR) and glucuronides (-GLUC) generated by cytochrome P450s (CYP) and UDP-glucuronosyltransferases (UGTs), respectively. Evaluation of quality control samples and three commercially available human samples showed that our analytical approach has the ability to measure 24 unique WAR metabolites in human urine. Evaluation of the human data also provides new insights for evaluating WAR toxicity and begins characterizing important UGT metabolic pathways responsible for WAR detoxification. Data revealed the significance of specific metabolites among patients and the corresponding enzymatic capacity to generate these compounds, including the first report of direct WAR glucuronidation. On the basis of total OH-WAR levels, (S)-7-OH-WAR was the predominant metabolite indicating the significance of CYP2C9 in WAR metabolism, although other CYP2C enzymes also contributed to clearance of this isomer. (R)-WAR hydroxylation to OH-WARs was more diverse among the patients as reflected in varying contributions of CYP1A2 and multiple CYP2C enzymes. There was wide variation in the glucuronidation of WAR and the OH-WARs with respect to the compounds and patients. 6- and 7-OH-WAR were primarily (>70%) excreted as glucuronides unlike 4'-OH-WAR and 8-OH-WAR. For all patients, UGT1A1 is likely responsible for 6-O-GLUC production, although UGT1A10 may also contribute in one patient. 7-O-GLUC levels reflected contributions from potentially five different UGT1A enzymes. In all cases, WAR, 4'-OH-WAR, 8-OH-WAR, and the corresponding glucuronides were minor metabolites with respect to the others. Taken together, these data suggest that both P450 and UGT reactions contribute to the generation of excretable products in human urine, thereby generating complex metabolic networks.

Synchronous-digitization for Video Rate Polarization Modulated Beam Scanning Second Harmonic Generation Microscopy.

Fast beam-scanning non-linear optical microscopy, coupled with fast (8 MHz) polarization modulation and analytical modeling have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and linear Stokes ellipsometry imaging at video rate (15 Hz). NOSE enables recovery of the complex-valued Jones tensor that describes the polarization-dependent observables, in contrast to polarimetry, in which the polarization stated of the exciting beam is recorded. Each data acquisition consists of 30 images (10 for each detector, with three detectors operating in parallel), each of which corresponds to polarization-dependent results. Processing of this image set by linear fitting contracts down each set of 10 images to a set of 5 parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the fundamental laser beam. Using these parameters, it is possible to recover the Jones tensor elements of the sample at video rate. Video rate imaging is enabled by performing synchronous digitization (SD), in which a PCIe digital oscilloscope card is synchronized to the laser (the laser is the master clock.) Fast polarization modulation was achieved by modulating an electro-optic modulator synchronously with the laser and digitizer, with a simple sine-wave at 1/10th the period of the laser, producing a repeating pattern of 10 polarization states. This approach was validated using Z-cut quartz, and NOSE microscopy was performed for micro-crystals of naproxen.

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 multi-channel 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.

Spectral X-Ray Diffraction using a 6 Megapixel Photon Counting Array Detector.

Pixel-array array detectors allow single-photon counting to be performed on a massively parallel scale, with several million counting circuits and detectors in the array. Because the number of photoelectrons produced at the detector surface depends on the photon energy, these detectors offer the possibility of spectral imaging. In this work, a statistical model of the instrument response is used to calibrate the detector on a per-pixel basis. In turn, the calibrated sensor was used to perform separation of dual-energy diffraction measurements into two monochromatic images. Targeting applications include multi-wavelength diffraction to aid in protein structure determination and X-ray diffraction imaging.

Synchronous digitization for high dynamic range lock-in amplification in beam-scanning microscopy.

Digital lock-in amplification (LIA) with synchronous digitization (SD) is shown to provide significant signal to noise (S/N) and linear dynamic range advantages in beam-scanning microscopy measurements using pulsed laser sources. Direct comparisons between SD-LIA and conventional LIA in homodyne second harmonic generation measurements resulted in S/N enhancements consistent with theoretical models. SD-LIA provided notably larger S/N enhancements in the limit of low light intensities, through the smooth transition between photon counting and signal averaging developed in previous work. Rapid beam scanning instrumentation with up to video rate acquisition speeds minimized photo-induced sample damage. The corresponding increased allowance for higher laser power without sample damage is advantageous for increasing the observed signal content.

Real-time dynamic range and signal to noise enhancement in beam-scanning microscopy by integration of sensor characteristics, data acquisition hardware, and statistical methods.

Despite the ubiquitous use of multi-photon and confocal microscopy measurements in biology, the core techniques typically suffer from fundamental compromises between signal to noise (S/N) and linear dynamic range (LDR). In this study, direct synchronous digitization of voltage transients coupled with statistical analysis is shown to allow S/N approaching the theoretical maximum throughout an LDR spanning more than 8 decades, limited only by the dark counts of the detector on the low end and by the intrinsic nonlinearities of the photomultiplier tube (PMT) detector on the high end. Synchronous digitization of each voltage transient represents a fundamental departure from established methods in confocal/multi-photon imaging, which are currently based on either photon counting or signal averaging. High information-density data acquisition (up to 3.2 GB/s of raw data) enables the smooth transition between the two modalities on a pixel-by-pixel basis and the ultimate writing of much smaller files (few kB/s). Modeling of the PMT response allows extraction of key sensor parameters from the histogram of voltage peak-heights. Applications in second harmonic generation (SHG) microscopy are described demonstrating S/N approaching the shot-noise limit of the detector over large dynamic ranges.