Imaging local electric fields produced upon synchrotron X-ray exposure

Abstract

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.synchrotron | EFISH | X-ray damage | piezoelectric | structural biology T he ability to determine atomic-resolution structures and the quality of resulting structures recovered by X-ray diffraction can be profoundly affected by X-ray induced damage during the diffraction data acquisition. At the energies typically used for protein structure determination, for every X-ray photon resulting in elastic scattering approximately 10-fold more photons result in inelastic scattering or absorption and deposition of energy into the crystal. The excess energy can perturb the molecules within a crystal through a variety of different mechanisms, typically resulting in the reduction of overall diffracted intensity (1). With biological macromolecules, such as proteins, this effect in combination with the initial inherent degree of ordering often limits the achievable resolution possible through signal averaging.X-ray induced radiation damage in protein crystals can manifest as two different types: global and specific (1). Specific damage occurs when irradiation causes specific chemical changes within the protein such as loss of side chains or disulfide bridge linkages (2, 3), damage to active sites (2), and damage to metal centers within the protein (1). Disulfide anion radicals, evidence of specific damage, have previously been observed by UV-vis microspectrophotometry after X-ray exposure for tens of milliseconds (4). Damage to nonspecific sites, known also as global damage, results in a decrease in the intensity of the high-angle spots with increasing X-ray dose, and t...

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy

“…At the end, a final image is obtained at the same half-wave plate and analyzer angles as the first image as a control for photobleaching or movement of the sample. The PIPO-SHG data is fit using a trust region algorithm in MATLAB (The Mathworks, Inc.) using Equation (1) which describes the SHG intensity ( I SHG ) as a function of laser linear polarization angle ( θ ′) and analyzer angle ( φ ′) from a sample under the assumption that the fibril is cylindrically symmetric and letting = as is typical for rod like structures [ 14 , 37 ] and neglecting potential contributions from local field effects such as from charge on the fibril or the coverslip [ 38 ]: Here A is a constant of proportionality between the SHG electric field and measured intensity, F is the noise floor, and the structural parameters ρ and κ are defined as: where α is the tilt angle of the fibril from the focal plane [ Z – X , see Figure 1(b) ], and is the second order optical electric susceptibility tensor of the collagen fibril which relates the induced polarization in a material to the applied electric fields. The laser is directed along Y in the laboratory coordinate system, XYZ , while the fibril is oriented along z in the fibril frame coordinate system, xyz , as shown in Figure 1(b) .…”

Materials characterization by synchrotron x-ray microprobes and nanoprobes

“…14 are performed with three different approaches depending on the energy and time values of the process. The x-ray inelastic scattering, photon absorption, XRF, and Auger relaxation processes and collisional ionization are usually simulated via a MC approach where, starting from a high number of primary x rays (typically 10 6 ), and using the known energy-dependent atomic photoabsorption and scattering cross sections (Henke, Gullikson, and Davis, 1993), the paths of primary and secondary photons and electrons are simulated in a probabilistic manner (London et al, 2001;Moukhametzianov et al, 2008;Dettmar et al, 2015;Torsello et al, 2018). MC simulations produce a quantitative space and time-dependent description of the distribution of the particles (photons, electrons, and ions) and ultimately a distribution of dose.…”