Practical crystallography with a transmission electron microscope.

内容摘要

Three-dimensional electron diffraction (3DED) is a powerful technique providing for crystal structure solutions of sub-micron sized crystals too small for structure determination via X-ray techniques. The entry requirement, however, of a transmission electron microscope (TEM) adapted with bespoke software for coordinated sample stage rotation and continuous electron diffraction data acquisition has generally inhibited the wider uptake of 3DED. To address this limitation, we present novel DigitalMicrograph script GiveMeED appropriate for controlled 3DED data acquisition. The collection of useable reflections beyond 0.8 Å makes 3DED crystallographic processing effectively routine, using standard software and workflows derived from single-crystal X-ray diffraction (SCXRD) techniques. A full experimental workflow for 3DED on a conventional TEM is described in practical terms, in combination with direct imaging, and energy dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS), for the return of comprehensive correlative descriptions of crystal morphologies and sample compositions, with due regard for the quantification of electron flux at each stage of the characterisation process. The accuracy and effectiveness of GiveMeED is demonstrated through structure solutions for case study paracetamol, copper(II) phthalocyanine, and percholorocoronene samples, characterised in their near-native states under controlled low dose conditions at either room or cryogenic temperatures, with determined unit cell parameters and atomic connectivity matching accepted literature X-ray structures for these compounds. To promote the wider adoption of 3DED, we make GiveMeED freely available for use and modification, in support of greater uptake and utilisation of structure solution procedures via electron diffraction. LAY DESCRIPTION: Crystallography is the study of crystal structure and properties via diffraction pattern analysis, generated transmitting short-wavelength radiation (X-rays, electrons) through molecular crystals. A key application is solving crystal structures, in fields such as drug discovery, protein structure investigation, and framework material synthesis. Three-dimensional electron diffraction (3DED) is a powerful technique for determining crystal structures, often performed using a transmission electron microscope (TEM), suited to analysis of sub-micron sized crystals that are too small for X-ray diffraction methods, providing crystallographic characterisation of materials otherwise be inaccessible to diffraction methods. In a 3DED experiment, a series of diffraction patterns are recorded from a crystal rotated under electron beam illumination while the microscope is in diffraction mode. The 2D diffraction patterns are used to reconstruct the 3D diffraction sphere, to which established crystallographic techniques are applied to solve the crystal structure. Correlative structural and chemical analysis is achieved by acquiring images and spectra from the same specimen volume. Broad adoption of 3DED has been limited by access to specialised knowledge and custom software needed to control the TEM during 3DED experiments, and existing software options may not be compatible with all instrument setups. In this work we present a novel DigitalMicrograph script, GiveMeED, designed to enable reproducible 3DED experiments with on compatible TEM systems without requiring extensive modifications or custom coding. We demonstrate the capabilities of GiveMeED by solving the structures of small molecules paracetamol, percholorocoronene, and copper(II)-phthalocyanine. We describe a comprehensive experimental workflow combining 3DED with TEM imaging and spectroscopy, care given to the precise control and quantification of electron fluence at each stage of the experimental workflow in order to limit electron beam damage and maintain the specimen at near-native state during the diffraction experiment. GiveMeED is available via GitHub for use and modification, enabling the microscopy community greater access to 3DED. In providing a robust, repeatable workflow based on open-source software, this work advances the routine use of electron diffraction on standard TEM hardware.